Deoxy-cytidine or uridine derivatives for use in cancer therapies

ABSTRACT

The present invention relates to a compound of Formula (I), or a stereoisomer, solvate, tautomer or pharmaceutically acceptable salt thereof, wherein X, W 1 , W 2 , Y, Z, R 1 , R 2  and R 3  are as defined in the disclosure herein, for use in therapy, particularly for use in the treatment of cancer. The present invention also relates to methods of treating cancer comprising the administration of a compound of Formula (I) to a subject in need thereof, and to pharmaceutical compositions and kits comprising such compounds.

The present invention relates to compounds for use in therapy,particularly in the treatment or prevention of cancer. The inventionalso relates to pharmaceutical formulations comprising such compoundsand to the use of such pharmaceutical formulations in therapy,particularly in the treatment or prevention of cancer. This inventionalso relates to such compounds for combined treatment with knownanticancer agents. Thus, the invention relates to the use in cancertreatment or prevention of the compounds of the invention both alone,and in combination with one or more additional or further anticanceragents. The invention also relates to methods of treating or preventingcancer using the compounds of the invention, either alone or incombination with one or more further anticancer agents.

Cancer is a disease characterized by the loss of appropriate control ofcell growth and proliferation. The American Cancer Society has estimatedthat there were in excess of 1.5 million new cases of cancer within theUnited States of America in 2010 and approximately 570,000 deaths thatyear estimated to be attributable to cancer. The World HealthOrganization has estimated that cancer was the leading cause of deathglobally in 2010, with the number of deaths caused by cancer growing to12 million per year by 2030.

Whilst there are numerous therapies available, resistance to knownanticancer drugs can be a problem in the successful treatment of cancerin patients. There remains a need for additional cancer therapies.

In some contexts, anticancer agents capable of killing a wide range ofcancer cell types are desirable. Fluorouracil (5-FU) is one suchanticancer agent, routinely administered for the treatment of a widerange of cancers.

However, many anticancer agents which are capable of killing a widerange of cancer cell types also kill non-cancerous cells, for instancenormal, healthy cells which are dividing. This is problematic in somecontexts, so there is also a need for broadly cytotoxic anticanceragents which are not cytotoxic against non-cancerous cells.Additionally, there is a need for anticancer agents with more specificcytotoxicity, i.e. which kill a smaller range of cancer cell types. Suchmore specific anticancer agents are less likely to have undesirableoff-target effects.

Glioblastoma multiforme is the most common and aggressive cancer of thecentral nervous system. Glioblastoma multiforme is the second mostcommon cancer in children. Adults diagnosed with glioblastoma multiformehave some of the highest unmet needs in oncology. With currentlyavailable treatments, mean survival from diagnosis is 14.6 months. Lessthan 5% of patients diagnosed with glioblastoma multiforme survivelonger than 5 years. Long-term survival after glioblastoma multiformediagnosis is rare and is more common in children.

Present therapies are largely palliative and designed to improve qualityof life. After diagnosis, current glioblastoma multiforme treatmentfollows a similar course: surgical resection of the affected brain area,where the patient is expected to survive the surgery, followed byradiation and chemotherapies. Glioblastoma multiforme forms tentaclelike structures in the afflicted brain; therefore, even where indicatedcomplete surgical resection is challenging and often impossible. Asidefrom surgical intervention and radiation therapy, three drugs areapproved for glioblastoma multiforme treatment:

1) Avastin® (bevacizumab)—an angiogenesis inhibitor with multipleoncology indications. Avastin® is thought to inhibit tumour growth byinhibiting the development of new blood vessels within tumours,effectively starving the tumour. Avastin® is approved in the USA and inJapan; however, Avastin® is not approved for GBM treatment in the EU.Indeed, Phase II trials indicated that Avastin® did not yield an overallsurvival benefit (NCI 06-C-0064E). While Avastin® slightly improvesprogression free survival in some trials, the patient benefit is oftennominal and there is no benefit afforded by Avastin® treatment inrecurrent glioblastoma multiforme patients.

2) Temozolomide(Temodal®/Temodar®)(4-methyl-5-oxo-2,3,4,6,8-pentazabicyclo[4.3.0]nona-2,7,9-triene-9-carboxamide)—amutagenic DNA alkylating agent; generic formulations are also available.Temozolomide is believed to inhibit glioblastoma multiforme growth byseverely mutating tumour DNA inducing cell death. Due to the mechanismof action, cells that express the DNA repair protein MGMT are almostuniversally resistant to temozolomide treatment. Temozolomide treatmentimproves overall survival by 2.5 months. At the same time, temozolomidetreatment often causes significant side-effects. Temozolomide is theprimary treatment for glioblastoma multiforme.

3) Gliadel® (Carmustine wafer)(1,3-Bis(2-chloroethyl)-1-nitrosourea)—acytotoxic nitrogen mustard. It is delivered as a biodegradable discimplanted directly into the brain after surgical resection. A randomizedtrial demonstrated that Gliadel® improves median survival by 2.1 months.Patients treated with Gliadel® report fewer and less severe side-effectsthan temozolomide treated patients; however, compared to temozolomideoverall survival is reduced.

There remains a need for further anticancer agents for the treatment ofglioblastoma multiforme and other cancers. In particular there remains aneed for anticancer agents which effectively treat cancers which areresistant to treatment with Temozolomide.

The human protein cytidine deaminase (CDA) catalyses hydrolyticdeamination of cytidine and deoxycytidine into uridine and deoxyuridine,respectively. Some known anticancer agents are nucleoside/nucleotideanalogs, for instance gemcitabine (2,2-difluorodeoxycytidine) andcytarabine (Ara-C, cytosine arabinoside). CDA problematicallyinactivates such anticancer agents, including gemcitabine andcytarabine. There remains a need for further anticancer agents which arenot inactivated by CDA.

The present inventors have found a selected class of compounds whichexhibit an activity in treating glioblastoma multiforme and a wide rangeof other cancers. Such compounds are suitable for inhibiting theproliferation of tumour cells in general and, in particular, thoseassociated with cancers of the brain, particularly glioblastomamultiforme.

In a first aspect, the invention provides a compound of Formula (I):

or a stereoisomer, solvate, tautomer or pharmaceutically acceptable saltthereof for use in therapy;wherein:

-   -   X is a group containing from 1 to 20 non-hydrogen atoms, which        contains at least one functional group selected from an        aldehyde, an alcohol, a protected alcohol, an ether, an ester        and a carboxylic acid, with the proviso that X is not —COOH;    -   W₁ and W₂ are each independently O, S or NH;    -   Y is H or a group containing from 1 to 15 non-hydrogen atoms;    -   Z is —OPG, —OR, or —N(R_(x)R_(y)), where R_(x), R_(y) and R_(z)        are independently H or a group containing from 1 to 10        non-hydrogen atoms;    -   R₁ is H or a group containing from 1 to 15 non-hydrogen atoms;    -   R₂ is H, —OH, —OPG, —F, —Cl, —Br, —I, or —N₃; and    -   R₃ is H, —F, —Cl, —Br, —I, or —N₃;        where PG is an alcohol protecting group, such as acetyl (Ac),        benzyl (Bn) or benzoyl (Bz).

The compounds of the invention are particularly for use in the treatmentor prevention of cancer. Thus, in a further aspect the present inventionprovides a compound of Formula (I):

or a stereoisomer, solvate, tautomer or pharmaceutically acceptable saltthereof for use in the treatment or prevention of cancer;wherein:

-   -   X is a group containing from 1 to 20 non-hydrogen atoms, which        contains at least one functional group selected from an        aldehyde, an alcohol, a protected alcohol, an ether, an ester        and a carboxylic acid, with the proviso that X is not —COOH;    -   W₁ and W₂ are each independently O, S or NH;    -   Y is H or a group containing from 1 to 15 non-hydrogen atoms;    -   Z is —OPG, —OR, or —N(R_(x)R_(y)), where R_(x), R_(y) and R_(z)        are independently H or a group containing from 1 to 10        non-hydrogen atoms;    -   R₁ is H or a group containing from 1 to 15 non-hydrogen atoms;    -   R₂ is H, —OH, —OPG, —F, —Cl, —Br, —I, or —N₃; and    -   R₃ is H, —F, —Cl, —Br, —I, or —N₃;        where PG is an alcohol protecting group, such as acetyl (Ac),        benzyl (Bn) or benzoyl (Bz).

The present invention is applicable to any cancer. Cancer is definedbroadly herein to include any neoplastic condition, and includesparticularly malignant or pre-malignant conditions. The cancer may causeor result in or manifest in solid tumours, but is not limited to such,and includes also cancers of the haempoietic system. Throughout, theterms “cancer” and “cancer cells” are used interchangeably. Throughout,the terms “tumour” and “tumour cells” are used interchangeably. Benigntumours and malignant tumours are also included in the term cancer asused herein, i.e. the terms cancer and tumour are used interchangeably.The treatment of malignant tumours is preferred.

Thus, alternatively viewed, the present invention provides a compound ofFormula (I) for use in the treatment or prevention of a tumour.

Alternatively viewed, the present invention provides a compound ofFormula (I) for use as an anticancer agent. Alternatively viewed, thepresent invention provides the use of a compound of Formula (I) fortreating or preventing cancer.

Alternatively viewed, the present invention provides a compound ofFormula (I) for use as an anti-tumour agent. Alternatively viewed, thepresent invention provides the use of a compound of Formula (I) fortreating or preventing a tumour.

Alternatively viewed, this aspect of the present invention provides useof a compound of Formula (I) in the manufacture of an anticancertherapeutic product (i.e. a preparation or medicament, for example apharmaceutical composition, formulation, combined product, co-formulatedproduct, or kit), or alternatively put, for the manufacture of amedicament for use as an anticancer agent or in the treatment orprevention of cancer.

Alternatively viewed, this aspect of the present invention provides useof a compound of Formula (I) in the manufacture of an anti-tumourtherapeutic product (i.e. a preparation or medicament, for example apharmaceutical composition, formulation, combined product, co-formulatedproduct, or kit), or alternatively put, for the manufacture of amedicament for use as an anti-tumour agent or in the treatment orprevention of a tumour.

In a further aspect, the present invention also provides a method oftreating or preventing cancer in a subject which method comprisesadministering a compound of Formula (I) to said subject.

In a further aspect, the present invention also provides a method oftreating or preventing a tumour in a subject which method comprisesadministering a compound of Formula (I) to said subject.

In some preferred embodiments of the present invention the compound ofFormula (I) is used as the sole active agent (sole active agent in thetreatment regimen). Thus, in some preferred embodiments the treatment isa monotherapy. Monotherapy refers to the use of a single drug to treat adisease or condition, in this cancer (i.e. a tumour). Thus, in somepreferred embodiments the compound of Formula (I) is used alone. By“sole active agent” (or sole active ingredient) is meant the sole agentor ingredient that is therapeutically active (or biologically active).Thus, components such as preservatives or excipients or agents that arenot relevant to the disease being treated are not considered to beactive agents.

In the treatment or prevention of cancer (i.e. a tumour) the compound ofFormula (I) may be used alone or optionally in combination with afurther, i.e. one or more other, anticancer agent(s) (i.e. an additionalor second anticancer agent(s)).

The compound of Formula (I), either alone or in combination with afurther anticancer agent, may be used according to the present inventionin any method of treatment or prevention of cancer (i.e. of a tumour) ina subject.

As demonstrated in the Examples, the use of a compound of Formula (I) incombination with one or more further anticancer agents results insynergistic cytotoxic effects.

Accordingly, in a further aspect, the present invention provides acompound of Formula (I) together with a further anticancer agent for usein treating or preventing cancer(i.e. a tumour), or alternatively put, acompound of Formula (I) for use together with a further anticancer agentfor treating or preventing cancer (i.e. a tumour).

Alternatively viewed, this aspect of the present invention provides useof a compound of Formula (I) in the manufacture of an anticancer (i.e.anti-tumour) therapeutic product (i.e. a preparation or medicament, forexample a pharmaceutical composition, formulation, combined product orkit), or alternatively put, for the manufacture of a medicament for useas an anticancer agent (i.e. anti-tumour agent) or in the treatment orprevention of cancer (i.e of a tumour), wherein said treatment furthercomprises the administration of a further anticancer agent.

In a further aspect, the present invention also provides a method oftreating or preventing cancer (i.e a tumour) in a subject, which methodcomprises administering a compound of Formula (I), optionally togetherwith a further (i.e. second) anticancer agent, to said subject.Particularly, in this aspect the method comprises administering aneffective amount of said compound of Formula (I) and the optionalfurther anticancer agent.

The compound of Formula (I) and the further anticancer agent(s) may beco-formulated into a single composition. However, this is not necessary.The medicament may be a combined preparation, composition or kit etc.and it is not necessary in any of the aspects of the invention for thecompound of Formula (I) and the further anticancer agent(s) to beco-formulated in a single composition—they may be separately formulatedand may be administered separately, including sequentially orsimultaneously.

Accordingly, the invention also provides a kit comprising a compound ofFormula (1) and a further (i.e. one or more further or second)anticancer agent(s) for using in the treatment or prevention of cancer(i.e. of a tumour).

More particularly, the invention provides a product (particularly apharmaceutical product) comprising a compound of Formula (I) and afurther (i.e. one or more further or second) anticancer agent(s) as acombined preparation for separate, sequential or simultaneous use in thetreatment or prevention of cancer. (i.e. of a tumour).

Additionally, the present invention provides a product (particularly apharmaceutical product) comprising a compound of Formula (I)co-formulated with a further (i.e. one or more further or second)anticancer agent(s).

The present invention provides a pharmaceutical composition comprising acompound of Formula (I) and one or more pharmaceutically acceptableexcipients, optionally further comprising a further anticancer agent.

In relation to all aspects of the invention, the compound of theinvention is the compound of Formula (I) as described elsewhere herein,and the preferred and optional embodiments concerning the compounddescribed in relation to one aspect of the invention apply mutatismutandis to each and every other aspect of the invention.

In all aspects and embodiments of the invention, the treatment ofmalignant tumours is preferred.

X

X is a group containing from 1 to 20 non-hydrogen atoms, which containsat least one functional group selected from an aldehyde, an alcohol, aprotected alcohol, an ether, an ester and a carboxylic acid, with theproviso that X is not —COOH.

Preferably, X is a group containing from 1 to 10 non-hydrogen atoms,more preferably from 1 to 5 non-hydrogen atoms, even more preferablyfrom 1 to 3 non-hydrogen atoms, and most preferably 2 non-hydrogenatoms.

More preferably, X is a group containing at least 2 non-hydrogen atoms,i.e. a group containing from 2 to 20 non-hydrogen atoms. Thus,preferably X is a group containing from 2 to 10 non-hydrogen atoms, evenmore preferably from 2 to 5 non-hydrogen atoms, even more preferablyfrom 2 to 3 non-hydrogen atoms, and most preferably 2 non-hydrogenatoms.

Preferably, X contains at least one functional group selected from analdehyde, an alcohol, a protected alcohol, an ether and an ester. Morepreferably, X contains at least one functional group selected from analdehyde, an alcohol, an ether and an ester. Most preferably, X containsat least one functional group selected from an aldehyde and an alcohol.For example X preferably contains an aldehyde functional group. Forexample X preferably contains an alcohol functional group.

Preferably, X contains just one functional group.

Preferably, X is a group as defined herein, with the proviso that X isnot —COOH or —OH.

X may be defined as -L-X′, wherein:

L is a bond, alkyl, alkenyl, alkynyl, haloalkyl, alkoxy, haloalkoxy; and

X′ is —CHO, —OH, —OPG, —COOH, —OR, —OC(═O)R or —C(═O)OR, wherein PG isan alcohol protecting group, such as acetyl (Ac), benzyl (Bn) or benzoyl(Bz), and wherein R is an alkyl group, preferably methyl.

The term “alkyl” refers to straight and branched saturated aliphatichydrocarbon chains. Preferably, alkyl refers to C₁₋₁₀ alkyl. Examplealkyl groups include, but are not limited to, methyl (Me), ethyl (Et),propyl (e.g., n-propyl and isopropyl), butyl (e.g., n-butyl, isobutyl,t-butyl), and pentyl (e.g., n-pentyl, isopentyl, neopentyl).

R may be any alkyl group, such those exemplified above. For example, Rmay be —(CH₂)_(n)H, where n is from 1 to 10, preferably from 1 to 5,more preferably from 1 to 3, and most preferably 1. When n is 1, R isCH₃.

The term “alkenyl” refers to straight and branched hydrocarbon chainshaving one or more, preferably one or two, carbon-carbon double bonds.Preferably, alkenyl refers to C₂₋₁₀ alkenyl. Examples of alkenyl groupsinclude, but are not limited to, ethenyl, 1-propenyl, 2-propenyl,2-butenyl, 3-butenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 2-hexenyl,3-hexenyl, 4-hexenyl, 5-hexenyl, 2-methyl-2-propenyl, and4-methyl-3-pentenyl.

The term “alkynyl” refers to straight and branched hydrocarbon chainshaving one or more, preferably one or two, carbon-carbon triple bonds.Preferably, alkynyl refers to C₂₋₁₀ alkynyl. Examples of alkynyl groupsinclude, but are not limited to, ethynyl, propynyl, and propargyl.

The term “haloalkyl” refers to straight and branched saturated aliphatichydrocarbon chains substituted with 1 or more halogens (fluoro (F),chloro (Cl), bromo (Br), and iodo (I)). Preferably, haloalkyl refers toC₁₋₁₀ haloalkyl. Examples of haloalkyl groups include, but are notlimited to, fluoromethyl, difluoromethyl, trifluoromethyl,trichloromethyl, pentafluoroethyl, pentachloroethyl,2,2,2-trifluoroethyl, heptafluoropropyl, and heptachloropropyl.

The term “alkoxy” refers to an —O-alkyl group. Preferably, alkoxy refersto C₁₋₁₀ alkoxy. Example alkoxy groups include, but are not limited to,methoxy, ethoxy, propoxy (e.g., n-propoxy and isopropoxy), and t-butoxy.

The term “haloalkoxy” refers to a haloalkyl group as defined aboveattached through an oxygen bridge. Preferably, haloalkoxy refers toC₁₋₁₀ haloalkoxy.

Examples of haloalkoxy groups include, but are not limited to,trifluoromethoxy, 2,2,2-trifluoroethoxy, and pentafluorothoxy.

When X is -L-X′, the X group must still contain the required number ofnon-hydrogen atoms.

Preferably, L is a bond, alkyl, alkenyl or alkynyl, more preferably abond or alkyl. For example, L may be a bond or C₁₋₆ alkyl. Morepreferably, L is a bond or C₁₋₄ alkyl. Most preferably, L is a bond orC₁ alkyl (—CH₂—).

X′ is preferably —CHO, —OH, —OPG, —OR, —OC(═O)R or —C(═O)OR, morepreferably —CHO, —OH, —OR, —OC(═O)R or —C(═O)OR, most preferably —CHO,—OH, —OR or —OC(═O)R.

Thus, preferably X is —(CH₂)_(n)—X′, wherein n is from 0 to 6,preferably from 0 to 4 and more preferably 0 or 1, and X is as definedabove, preferably —CHO, —OH, —OR or —OC(═O)R, where R is as definedabove.

Preferably X is —(CH₂)_(n)—X′, wherein n is from 0 to 6 and X is —OH or—CHO. Preferably X is —(CH₂)_(n)—X′, wherein n is from 0 to 6 and X′ is—OH. Preferably X is —(CH₂)_(n)—X′, wherein n is from 0 to 6 and X is—CHO.

More preferably X is —(CH₂)_(n)—X′, wherein n is from 0 to 4 and X is—OH or —CHO. Preferably X is —(CH₂)_(n)—X′, wherein n is from 0 to 4 andX′ is —OH. Preferably X is —(CH₂)_(n)—X′, wherein n is from 0 to 4 and Xis —CHO.

More preferably X is —(CH₂)_(n)—X′, wherein n is from 0 to 2 and X is—OH or —CHO. Preferably X is —(CH₂)_(n)—X′, wherein n is from 0 to 2 andX′ is —OH. Preferably X is —(CH₂)_(n)—X′, wherein n is from 0 to 2 and Xis —CHO.

More preferably X is —(CH₂)_(n)—X′, wherein n is 0 or 1 and X is —OH or—CHO. Preferably X is —(CH₂)_(n)—X′, wherein n is 0 or 1 and X′ is —OH.Preferably X is —(CH₂)_(n)—X′, wherein n is 0 or 1 and X is —CHO.

More preferably, X is —CHO, —CH₂OH, —CH₂OCH₃ or —CH₂OC(═O)CH₃.

More preferably, X is a) —CHO or —CH₂OH; or b) —CH₂OCH₃ or—CH₂OC(═O)CH₃.

More preferably X is —CHO or —CH₂OH, most preferably CH₂OH.

In all of the above definitions of X, it is preferred that X is not —OH.Thus, when X′ is —OH, it is preferred that L is not a bond (i.e. n isnot 0). In this case, n may be from 1 to 6, preferably from 1 to 4, morepreferably from 1 to 2, and most preferably 1.

W₁ and W₂

W₁ and W₂ are each independently O, S or NH, preferably O or S, morepreferably O.

Thus, preferably W₁ is O or S and W₂ is O, S or NH; or W₂ is O or S andW₁ is O, S or NH.

More preferably, W₁ and W₂ are both O or S, and even more preferably W₁is O and W₂ is O or S; or W₂ is O, and W₁ is O or S.

Most preferably, W₁ and W₂ are both O.

Y

Y is H or a group containing from 1 to 15 non-hydrogen atoms.Preferably, Y is H or a group containing from 1 to 10 non-hydrogenatoms. More preferably, Y is H or a group containing from 1 to 5non-hydrogen atoms.

For example, Y may be H, —OH, —OPG, —F, —Cl, —Br, —I, or —N₃, where PGis an alcohol protecting group, such as acetyl, benzyl or benzoyl.

When Y is H or a group containing from 1 to 5 non-hydrogen atoms, Y maybe H, —OH, —OAc, —F, —Cl, —Br, —I, or —N₃.

Most preferably, Y is H.

Z

Z is —OPG, —OR, or —N(R_(x)R_(y)), where Rx, R_(y) and R_(z) areindependently H or a group containing from 1 to 10 non-hydrogen atoms,and where PG is an alcohol protecting group, such as acetyl, benzyl orbenzoyl.

Preferably, Z is —OR_(z) or —N(R_(x)R_(y)).

Preferably, R_(z) is H or a group containing from 1 to 5 non-hydrogenatoms, more preferably H or a group containing from 1 to 3 non-hydrogenatoms, and most preferably H.

Preferably, R_(x) and R_(y) are independently H or a C₁₋₈ ester. Morepreferably, RX and R_(y) are independently H or —C(O)O(CH₂)_(n)CH₃,where n is from 1 to 4, preferably 4.

Preferably, at least one of R, and R_(y) are H. For example, preferablyR, is H and R_(y) is independently H or —C(O)O(CH₂)_(n)CH₃, where n isfrom 1 to 4, preferably 4. More preferably, R, and R_(y) are both H.

Z is therefore preferably —NH₂ or —OH. More preferably, Z is —NH₂ when Xis —CHO or —CH₂OH, most preferably —CH₂OH; and Z is —OH when X is—CH₂OCH₃ or —CH₂OC(═O)CH₃.

When Z is —OH, the compound of formula (I) can be drawn in a tautomericform, as shown below:

-   -   A tautomeric form of Formula (I), when Z is —OH

Alternatively, Z is preferably —OPG, —OR_(z) or —N(R_(x)R_(y)), wherePG, R_(x) and R_(y) are as defined above, and where R_(z) is a groupcontaining from 1 to 10 non-hydrogen atoms.

In this case, R_(z) is preferably a group containing from 1 to 5non-hydrogen atoms, more preferably a group containing from 1 to 3non-hydrogen atoms.

In this case, Z is preferably —OPG or —N(R_(x)R_(y)), wherein PG, R_(x)and R_(y) are as defined above.

Most preferably, Z is —N(R_(x)R_(y)), wherein R_(x) and R_(y) are asdefined above.

Z is therefore preferably —NH₂.

R₁

R₁ is H or a group containing from 1 to 15 non-hydrogen atoms,preferably H or a group containing from 1 to 13 non-hydrogen atoms.

Preferably, R₁ is H, —OH, —OPG, —F, —Cl, —Br, —I, —N₃, or—O(P(=O)(OH)O)_(n)H, where n is from 1 to 3, and where PG is an alcoholprotecting group, such as acetyl, benzyl or benzoyl.

Preferably, R₁ is H, —OH, —F, —Cl, —Br, —I, —N₃, or —O(P(=O)(OH)O)_(n)H,where n is from 1 to 3, preferably 3. More preferably, R, is H, —OH or—O(P(=O)(OH)O)_(n)H, where n is from 1 to 3, preferably 3. Even morepreferably, R, is —OH or —O(P(=O)(OH)O)_(n)H, where n is from 1 to 3,preferably 3. Most preferably R, is —OH.

R₂

R₂ is H, —OH, —OPG, —F, —Cl, —Br, —I, or —N₃, where PG is an alcoholprotecting group, such as acetyl, benzyl or benzoyl.

Preferably, R₂ is H, —OH, —F, —Cl, —Br, —I, or —N₃. More preferably, R₂is H or —OH, and most preferably R₂ is —OH.

R₃

R₃ is H, —F, —Cl, —Br, —I, or —N₃, preferably H.

Most preferably, R, is —OH or —O(P(=O)(OH)O)_(n)H where n is from 1 to3, preferably 3; R₂ is —OH; and R₃ is H.

PREFERRED EMBODIMENTS

Preferably, the compound of Formula (I) is a compound of Formula (IIa)or Formula (IIb), or a stereoisomer, solvate, tautomer orpharmaceutically acceptable salt thereof:

wherein X, R₁ and R₂ are as defined above.

More preferably, the compound of Formula (I) is a compound of Formula(IIa), or a stereoisomer, solvate, tautomer or pharmaceuticallyacceptable salt thereof.

In these preferred embodiments, particularly in Formula (IIa),preferably X is —(CH₂)_(n)—X′, wherein n is from 0 to 6 and X is —OH or—CHO. Preferably X is —(CH₂)_(n)—X′, wherein n is from 0 to 6 and X′ is—OH. Preferably X is —(CH₂)_(n)—X′, wherein n is from 0 to 6 and X is—CHO.

More preferably X is —(CH₂)_(n)—X′, wherein n is from 0 to 4 and X is—OH or —CHO. Preferably X is —(CH₂)_(n)—X′, wherein n is from 0 to 4 andX′ is —OH. Preferably X is —(CH₂)_(n)—X′, wherein n is from 0 to 4 and Xis —CHO.

More preferably X is —(CH₂)_(n)—X′, wherein n is from 0 to 2 and X is—OH or —CHO. Preferably X is —(CH₂)_(n)—X′, wherein n is from 0 to 2 andX′ is —OH. Preferably X is —(CH₂)_(n)—X′, wherein n is from 0 to 2 and Xis —CHO.

More preferably X is —(CH₂)_(n)—X′, wherein n is 0 or 1 and X is —OH or—CHO. Preferably X is —(CH₂)_(n)—X′, wherein n is 0 or 1 and X is —OH.Preferably X is —(CH₂)_(n)—X′, wherein n is 0 or 1 and X is —CHO.

More preferably X is —CHO, —CH₂OH, —CH₂OCH₃ or —CH₂OC(═O)CH₃. Morepreferably, X is a) —CHO or —CH₂OH; or b) —CH₂OCH₃ or —CH₂OC(═O)CH₃.More preferably X is —CHO or —CH₂OH, most preferably CH₂OH

In Formula (IIa), preferably X is —CHO or —CH₂OH, most preferably—CH₂OH. In Formula (IIb), preferably X is —CH₂OCH₃ or —CH₂OC(═O)CH₃.

In all of these preferred embodiments, it is preferred that X is not—OH. Thus, when X′ is —OH, it is preferred that. n is not 0. In thiscase, n may be from 1 to 6, preferably from 1 to 4, more preferably from1 to 2, and most preferably 1.

In Formula (IIa) and (IIb), preferably R, is —OH or —O(P(=O)(OH)O)_(n)Hwhere n is from 1 to 3, preferably 3; and R₂ is —OH.

More preferably, the compound of Formula (I) is a compound of Formula(IIIa), (IIIb), (IIIc) or (IIId) or a stereoisomer, solvate, tautomer orpharmaceutically acceptable salt thereof:

wherein X is as defined above.

More preferably, the compound of Formula (I) is a compound of Formula(IIIa), (IIIb) or (IIIc) or a stereoisomer, solvate, tautomer orpharmaceutically acceptable salt thereof.

Even more preferably, the compound of Formula (I) is a compound ofFormula (IIIa) or (IIIc), or a stereoisomer, solvate, tautomer orpharmaceutically acceptable salt thereof.

In Formula (IIIa), (IIIb), (IIIc) and (IIId), particularly in Formula(IIIa) and (IIIc), preferably X is —(CH₂)_(n)—X′, wherein n is from 0 to6 and X is —OH or —CHO.

Preferably X is —(CH₂)_(n)—X′, wherein n is from 0 to 6 and X′ is —OH.Preferably X is —(CH₂)_(n)—X′, wherein n is from 0 to 6 and X is —CHO.

More preferably X is —(CH₂)_(n)—X′, wherein n is from 0 to 4 and X is—OH or —CHO.

Preferably X is —(CH₂)_(n)—X′, wherein n is from 0 to 4 and X′ is —OH.Preferably X is —(CH₂)_(n)—X′, wherein n is from 0 to 4 and X is —CHO.

More preferably X is —(CH₂)_(n)—X′, wherein n is from 0 to 2 and X is—OH or —CHO.

Preferably X is —(CH₂)_(n)—X′, wherein n is from 0 to 2 and X′ is —OH.Preferably X is —(CH₂)_(n)—X′, wherein n is from 0 to 2 and X is —CHO.

More preferably X is —(CH₂)_(n)—X′, wherein n is 0 or 1 and X is —OH or—CHO. Preferably X is —(CH₂)_(n)—X′, wherein n is 0 or 1 and X is —OH.Preferably X is —(CH₂)_(n)—X′, wherein n is 0 or 1 and X is —CHO.

More preferably X is —CHO, —CH₂OH, —CH₂OCH₃ or —CH₂OC(═O)CH₃. Morepreferably, X is a) —CHO or —CH₂OH; or b) —CH₂OCH₃ or —CH₂OC(═O)CH₃.More preferably X is —CHO or —CH₂OH, most preferably CH₂OH

In Formula (IIIa) and Formula (IIIc), preferably X is —CHO or —CH₂OH,most preferably —CH₂OH. In Formula (IIIb) and Formula (IIId), preferablyX is —CH₂OCH₃ or —CH₂OC(═O)CH₃.

In all of these preferred embodiments, it is preferred that X is not—OH. Thus, when X′ is —OH, it is preferred that. n is not 0. In thiscase, n may be from 1 to 6, preferably from 1 to 4, more preferably from1 to 2, and most preferably 1.

Most preferably, the compound of Formula (I) is a compound of Formula(IVa), (IVb), (IVc), (IVd), (IVe) or (IVf) or a stereoisomer, solvate,tautomer or pharmaceutically acceptable salt thereof:

Formula (IVa) is 5-formyl-2′-deoxycytidine (also termed 5f2dC, 5fdC,2d5fC and d5fC herein). Formula (IVb) is5-hydroxymethyl-2′-deoxycytidine (also termed 5hm2dC, 5hmdC, 2d5hmC andd5hmC herein). Formula (IVc) is 5-methoxymethyl-2′-deoxyuridine. Formula(IVd) is 5-acetoxymethyl-2′-deoxyuridine Formula (IVe) is5-formyl-2′-deoxycytidine-5′-triphosphate. Formula (IVf) is5-hydroxymethyl-2′-deoxycytidine-5′-triphosphate.

Thus, preferably, the compound of use in the invention is selected fromis 5-formyl-2′-deoxycytidine, 5-hydroxymethyl-2′-deoxycytidine,5-methoxymethyl-2′-deoxyuridine, 5-acetoxymethyl-2′-deoxyuridine,5-formyl-2′-deoxycytidine-5′-triphosphate and5-hydroxymethyl-2′-deoxycytidine-5′-triphosphate or a stereoisomer,solvate, tautomer or pharmaceutically acceptable salt thereof. Morepreferably the compound is a) 5-formyl-2′-deoxycytidine or5-hydroxymethyl-2′-deoxycytidine or a stereoisomer, solvate, tautomer orpharmaceutically acceptable salt thereof; or b)5-methoxymethyl-2′-deoxyuridine or 5-acetoxymethyl-2′-deoxyuridine or astereoisomer, solvate, tautomer or pharmaceutically acceptable saltthereof

Alternatively, the compound of use in the invention is preferablyselected from 5-formyl-2′-deoxycytidine,5-hydroxymethyl-2′-deoxycytidine,5-formyl-2′-deoxycytidine-5′-triphosphate and5-hydroxymethyl-2′-deoxycytidine-5′-triphosphate or a stereoisomer,solvate, tautomer or pharmaceutically acceptable salt thereof.

Most preferably, the compound is 5-formyl-2′-deoxycytidine or5-hydroxymethyl-2′-deoxycytidine or a stereoisomer, solvate, tautomer orpharmaceutically acceptable salt thereof, most preferably5-hydroxymethyl-2′-deoxycytidine or a stereoisomer, solvate, tautomer orpharmaceutically acceptable salt thereof.

The term “treatment” or “therapy” includes any treatment or therapywhich results in an improvement in the health or condition of a patient,or of a symptom of the cancer they are suffering. “Treatment” is notlimited to curative therapies (e.g. those which result in theelimination of cancer cells or tumours or metastases from the patient),but includes any therapy which has a beneficial effect on the cancer orthe patient, for example, tumour regression or reduction, reduction ofmetastatic potential, increased overall survival, extension orprolongation of life or remission, induction of remission, a slow-downor reduction of disease progression or the rate of disease progression,or of tumour development, subjective improvement in quality of life,reduced pain or other symptoms related to the disease, improvedappetite, reduced nausea, or an alleviation of any symptom of thecancer.

Thus, as used herein ‘treatment’ may refer to reducing, alleviating,ameliorating or eliminating the cancer, or one or more symptoms thereof,which is being treated, relative to the cancer or symptom prior to thetreatment. Treatment may include a reduction or elimination of cancercells, for example in tumours, e.g. in solid tumours. Treatment istreatment of a subject, i.e. a subject in need thereof. Thus, treatmentmay include a reduction in tumour size, or the prevention of tumourgrowth or further tumour growth, i.e. stabilization of tumour size.

“Prevention” refers to delaying or preventing the onset of the symptomsof the cancer, e.g. in the development of a tumour.

Preferably, the compounds of the invention have a direct effect oncancer/tumour cells. A “direct effect” as used herein means that thecompounds of the invention interact directly with cancer/tumour cells inorder to exert their anti-cancer/anti-tumour effects. In other words,preferably the compounds of the invention, i.e. the compounds of Formula(I) are cytotoxic to cancer/tumour cells. Preferably, the compounds ofthe invention are administered to a subject in order to exert a directeffect against cancer/tumour cells.

Preferably, the methods of the invention do not comprise administrationof the compounds of the invention in order to deplete a population ofcells which has been administered as part of a cell-based therapy. Cellbased therapies are well-known as therapies in which a population ofcells is administered to a subject in order to elicit a particulartherapeutic effect. Well-known cell-based therapies include T-celltherapy, e.g. CAR T-cell therapy.

Preferably, the methods of the invention do not comprise administrationof the compounds of the invention after administration of a populationof cells which has been administered as part of a cell-based therapy.

Preferably, the methods of the invention do not comprise CAR T-celltherapy. Preferably, the methods of the invention do not comprise T-celltherapy. Preferably the methods of the invention do not comprisecell-based therapies.

Preferably the methods of the invention comprise administration of thecompound of the invention to a subject not undergoing CAR T-celltherapy, preferably T-cell therapy, preferably cell-based therapy. Inother words, preferably the subject has not received and will notreceive CAR-T cell therapy, preferably T-cell therapy, preferablycell-based therapy as part of the their treatment.

As referred to herein a subject may be any human or non-human animal,preferably a mammalian animal, e.g. a cow, horse, sheep, pig, goat,rabbit, cat, dog, especially preferably a human. Thus, preferably thecancers referred to herein are human cancers, and the tumours referredto herein are preferably present in a human subject.

In some embodiments, treatments in accordance with the present inventionmay be used in subjects at risk of cancer relapse or recurrence ormetastasis. Thus, alternatively viewed, the compounds of Formula (I) maybe used in the prevention of cancer relapse or recurrence or metastasis.

In particular embodiments the invention may involve first identifying ordetermining that the subject to be treated has cancer (i.e. a tumour) oris susceptible to or at risk of developing cancer.

Alternatively or additionally, the invention may involve assessing ormonitoring the effect of the administration of the compound of Formula(I) and/or the other anticancer agent(s) on the subject, or moreparticularly on the cancer (tumour), or on the development or progressof the cancer (tumour). Procedures and means for assessing and/ormonitoring an anticancer effect are well known in the art, for exampleby determining or monitoring symptoms, clinical condition, tumour sizeor spread (e.g. by imaging techniques) or other cancer or tumourindicators e.g. cancer/tumour markers etc.

As mentioned above, the present invention is applicable to any cancer.Cancer is defined broadly herein to include any neoplastic condition,and includes particularly malignant or pre-malignant conditions. Thecancer may cause or result in or manifest in solid tumours, but is notlimited to such, and includes also cancers of the haempoietic system.Benign tumours and malignant tumours are also included in the termcancer as used herein, i.e. the terms cancer and tumour are usedinterchangeably. The treatment of malignant tumours is preferred.

The cancer/may occur in any tissue or organ of the body. For example,the present invention can be used in the treatment or prevention of anyof the following cancers in a patient or subject:

Cancer of the Central Nervous System, preferably Brain Cancer,preferably Glioma; Acute Lymphoblastic Leukaemia (ALL); Acute MyeloidLeukaemia (AML); Adrenocortical Carcinoma; AIDS-Related Cancer (e.g.Kaposi Sarcoma and Lymphoma); Anal Cancer; Appendix Cancer; Basal CellCarcinoma; Bile Duct Cancer; Extrahepatic Bladder Cancer; Bone Cancer(e.g. Ewing Sarcoma; Osteosarcoma and Malignant Fibrous Histiocytoma);Breast Cancer; Bronchial Tumours; Burkitt Lymphoma; Carcinoid Tumour;Cardiac (Heart) Tumours; Cervical Cancer (Cervical Adenocarcinoma);Chordoma; Acute Promyelocytic Leukemia; Chronic Lymphocytic Leukemia(CLL); Chronic Myelogenous Leukaemia (CML); Chronic MyeloproliferativeDisorder; Colon Cancer; Colorectal Cancer; Cutaneous T-Cell Lymphoma;Bile Duct Cancer; Extrahepatic Ductal Carcinoma In Situ (DCIS);Embryonal Tumours; Endometrial Cancer; Esophageal Cancer;Esthesioneuroblastoma; Ewing Sarcoma; Extracranial Germ Cell Tumour;Extragonadal Germ Cell Tumour; Extrahepatic Bile Duct Cancer; Eye Cancer(including Intraocular Melanoma and Retinoblastoma); FibrousHistiocytoma of Bone; Gallbladder Cancer; Gastric (Stomach) Cancer;Gastrointestinal Carcinoid Tumour; Gastrointestinal Stromal Tumours(GIST); Germ Cell Tumor; Gestational Trophoblastic Disease; Hairy CellLeukaemia; Head and Neck Cancer; Heart Cancer; Hepatocellular (Liver)Cancer; Histiocytosis; Langerhans Cell; Hodgkin Lymphoma; HypopharyngealCancer; Intraocular Melanoma; Islet Cell Tumours; PancreaticNeuroendocrine Tumours; Kaposi Sarcoma; Kidney Cancer (including RenalCell and Wilms Tumour); Langerhans Cell Histiocytosis; Laryngeal Cancer;Leukaemia (including Acute Lymphoblastic (ALL); Acute Myeloid (AML);Chronic Lymphocytic (CLL); Chronic Myelogenous (CML); Lip and OralCavity Cancer; Liver Cancer (Primary); Lobular Carcinoma In Situ (LCIS);Lung Cancer; Lymphoma; Macroglobulinemia; Waldenström; Melanoma(Malignant Melanoma); Merkel Cell Carcinoma; Mesothelioma; MetastaticSquamous Neck Cancer with Occult Primary; Midline Tract CarcinomaInvolving NUT Gene; Mouth Cancer; Multiple Endocrine NeoplasiaSyndromes; Childhood; Multiple Myeloma/Plasma Cell Neoplasm; MycosisFungoides; Myelodysplastic Syndromes; Myelodysplastic/MyeloproliferativeNeoplasms; Multiple Myeloma; Myeloproliferative Disorders; Nasal Cavityand Paranasal Sinus Cancer; Nasopharyngeal Cancer; Neuroblastoma;Non-Hodgkin Lymphoma; Non-Small Cell Lung Cancer; Oral Cancer; OralCavity Cancer; Oropharyngeal Cancer; Osteosarcoma; Ovarian Cancer(Ovarian Adenocarcinoma); Pancreatic Cancer; Pancreatic NeuroendocrineTumours (Islet Cell Tumors); Papillomatosis; Paraganglioma; ParanasalSinus and Nasal Cavity Cancer; Parathyroid Cancer; Penile Cancer;Pharyngeal Cancer; Pheochromocytoma; Epithelial Adenocarcinoma; PlasmaCell Neoplasm/Multiple Myeloma; Pleuropulmonary Blastoma; Pregnancy andBreast Cancer; Prostate Cancer; Rectal Cancer; Renal Cell (Kidney)Cancer; Renal Pelvis and Ureter; Transitional Cell Cancer;Retinoblastoma; Rhabdomyosarcoma; Salivary Gland Cancer; Sarcoma; SézarySyndrome; Skin Cancer; Small Cell Lung Cancer; Small Intestine Cancer;Soft Tissue Sarcoma; Squamous Cell Carcinoma; Squamous Neck Cancer withOccult Primary; Metastatic; Stomach (Gastric) Cancer; T-Cell Lymphoma;Testicular Cancer; Throat Cancer; Thymoma and Thymic Carcinoma; ThyroidCancer; Transitional Cell Cancer of the Renal Pelvis and Ureter;Urethral Cancer; Uterine Cancer; Endometrial; Uterine Sarcoma; VaginalCancer; Vulvar Cancer; Waldenström Macroglobulinemia; and Wilms Tumour.

The cells of a human embryo are arranged into distinct germ layers: anouter ectoderm an inner endoderm, and the mesoderm, which developsbetween the ectoderm and the endoderm. All the organs of the bodydevelop or differentiate in an orderly fashion from these three primarygerm layers.

In the present invention, preferably the cancer/tumour is acancer/tumour of a tissue derived from the ectoderm, paraxial mesoderm,or lateral plate mesoderm, preferably from the ectoderm.

Preferably, the cancer is a cancer of the central nervous system,preferably brain cancer. For the avoidance of doubt, brain cancer isconsidered in the field and herein to be a cancer of the central nervoussystem. The central nervous system comprises the brain and the spinalcord. Thus, preferably the tumour is a tumour of the central nervoussystem, preferably a brain tumour. Preferably, the cancers/tumour of theCNS is selected from the group consisting of CNS lymphoma, RhabdoidTumour, Embryonal Tumours, Germ Cell Tumour and Chordoma, or is a braincancer/tumour. Preferably, the brain cancer/tumour is selected from thegroup consisting of Glioma, Acoustic Neuroma, CNS Lymphoma,Craniopharyngioma, Medulloblastoma, Meningioma, Metastatic Brain Tumor,Pituitary Tumors, Primitive Neuroectodermal (PNET), Schwannoma, PinealTumor, Trilateral Retinoblastoma and Rhabdoid Tumor.

Most preferably, the cancer/tumour is brain cancer/a brain tumour, morepreferably glioma. The glioma may be any type of glioma, for instanceastrocytoma, ependymoma, subependymoma, oligodendroglioma, brainstemglioma, optic nerve glioma or a mixed glioma.

Preferably, the glioma is astrocytoma. The astrocytoma may be Grade IAstrocytoma (preferably Pilocytic Astrocytoma or Subependymal giant cellastrocytoma), Grade II (preferably Low-grade Astrocytoma, Pleomorphicxanthoastrocytoma or Mixed oligoastrocytoma), Grade III (AnaplasticAstrocytoma) or most preferably Grade IV (Glioblastoma).

Grading systems for the classification of tumor of the central nervoussystem are well-known to the person of ordinary skill in the field.Preferably, the World Health Organization (WHO) grading system is used.The WHO grading scheme is well-known in the field and is based on theappearance of certain characteristics: atypia, mitosis, endothelialproliferation, and necrosis, which reflect the malignant potential ofthe tumor in terms of invasion and growth rate.

Gliomas may also be classified according to whether they are above orbelow the tentorium; a membrane which separates the cerebrum from thecerebellum. Supratentorial gliomas are found above the tentorium, in thecerebrum, whilst infratentorial gliomas are found below the tentorium,in the cerebellum. The glioma treated according to the present inventionmay be supratentorial glioma or infratentorial glioma.

Thus, particularly preferably in the context of the present invention,the cancer/tumour is glioma, most preferably Glioma, Grade IV, i.e.glioblastoma multiforme. Glioblastoma multiforme is a malignantastrocytoma and the most common primary brain tumor among adults.Glioblastoma multiforme is also known as Glioma, Grade IV, glioblastomaand GBM.

Preferably the cancer/tumour is selected from the group consisting ofbrain cancer (as defined above, preferably glioma, more preferablyglioblastoma), stomach cancer, pancreatic cancer, lymphoma, lung cancer,skin cancer, acute promyelocytic leukaemia, ovarian cancer, breastcancer, bone cancer and cervical cancer.

More preferably, the cancer/tumour is selected from the group consistingof brain cancer (as defined above, preferably glioma, more preferablyglioblastoma), stomach cancer, lymphoma, breast cancer and cervicalcancer.

Preferably, the cancer/tumour is selected from the group consisting ofbrain cancer (as defined above, preferably glioma, more preferablyglioblastoma), stomach cancer, pancreatic cancer, skin cancer, acutepromyelocytic leukaemia, breast cancer and cervical cancer.

Preferably, the cancer/tumour is selected from the group consisting ofbrain cancer (as defined above, preferably glioma, more preferablyglioblastoma), skin cancer and breast cancer, more preferably braincancer (as defined above, preferably glioma, more preferablyglioblastoma) and skin cancer.

In these embodiments, the preferred compounds of the invention are thosein which X contains an aldehyde functional group, preferably wherein Xis —(CH₂)_(n)—X′, wherein X′ is —CHO and n is from 0 to 6, morepreferably from 0 to 4, more preferably from 0 to 2, more preferably 0or 1, most preferably wherein X is —CHO, and preferably wherein Z is—NH₂. As demonstrated in the present Examples, such compounds of theinvention have advantageously broad cytotoxicity against a wide range ofcancer types, with limited cytotoxicity against non-cancerous cells.

Alternatively preferably the cancer/tumour is selected from the groupconsisting of brain cancer (as defined above, preferably glioma, morepreferably glioblastoma) and chronic chronic myelogenous leukemia. Inthese embodiments, the preferred compounds of the invention are those inwhich X contains an alcohol functional group, preferably wherein X is—(CH₂)_(n)—X′, wherein X′ is —OH and n is from 0 to 6, more preferablyfrom 0 to 4, more preferably from 0 to 2, more preferably 0 or 1, mostpreferably wherein X is —CH₂OH and preferably wherein Z is —NH₂. Asdemonstrated in the present Examples, such compounds of the inventionhave advantageously specific cytotoxicity against these preferred cancertypes, with limited cytotoxicity against off-target cancerous andnon-cancerous cells.

Preferably the stomach tumour is gastric carcinoma. Preferably thepancreatic tumour is pancreatic carcinoma. Preferably, the skin canceris malignant melanoma. Preferably the ovarian cancer is ovarianadenocarcinoma. Preferably the breast cancer is epithelialadenocarcinoma. Preferably the bone cancer is bone osteosarcoma.Preferably the lung cancer is metastatic adenocarcinoma, preferablymetastatic non-small cell adenocarcinoma. Preferably the cervical canceris cervical adenocarcinoma.

However, preferably the cancer is not lung cancer or breast cancer.Preferably the cancer is also not pancreatic cancer, stomach cancer,testis cancer or vaginal cancer. Preferably the cancer is also notkidney cancer or cancer of the intestine.

Preferably the cancer is not lung cancer. Preferably the cancer is alsonot prostate cancer, kidney cancer, liver cancer, breast cancer, coloncancer, ovarian cancer or cervical cancer.

Preferably the cancer/tumour is not colon cancer, lung cancer, prostatecancer or kidney cancer. Preferably the cancer/tumour is also notpancreatic cancer.

Preferably the cancer/tumour is also not chronic myelogenous leukaemia,and in this instance the preferred compounds of the invention (i.e. thecompounds of Formula (I), (IIa), (IIb), (IIIa), (IIIb), (IIIc), (IIId),(Iva), (IVb), (IVc), (IVd), (IVe) and(IVf)) are those in which Xcontains an aldehyde functional group, preferably wherein X is—(CH₂)_(n)—X′, wherein X is —CHO and n is from 0 to 6, more preferablyfrom 0 to 4, more preferably from 0 to 2, more preferably 0 or 1, mostpreferably wherein X is —CHO and preferably wherein Z is —NH₂.

Furthermore, preferably the cancer is also not:

-   -   i) breast cancer, preferably also not pancreatic cancer, stomach        cancer, testis cancer or vaginal cancer, more preferably also        not cancer of the intestine; and/or    -   ii) liver cancer, breast cancer, ovarian cancer or cervical        cancer.

Preferably the cancer is not a cancer selected from the group consistingof lung cancer, prostate cancer, kidney cancer, liver cancer, breastcancer, colon cancer, ovarian cancer, cervical cancer, chronicmyelogenous leukemia, pancreatic cancer, stomach cancer, testis cancer,vaginal cancer and cancer of the intestine. Preferably the cancer is notany of these cancers.

Preferably, the breast cancer treated according to the present invention

-   -   i) is invasive ductal carcinoma; and/or    -   ii) expresses wildtype p53; and/or    -   iii) is not triple negative, i.e. expresses one or more of        oestrogen receptor (ER+) progesterone receptor (PR+) and HER2        (HER2+), preferably is ER+ and PR+; and/or    -   iv) is heterozygous for p53.

The breast cancers preferably not treated according to the presentinvention are preferably only those breast cancer which:

-   -   i) are adenocarcinomas; and/or    -   ii) are triple negative, i.e. do not express oestrogen receptor        (ER−) progesterone receptor (PR−) or HER2 (HER2−); and/or    -   iii) express a mutant variant of p53; and/or    -   iv) are homozygous for p53.

The human protein cytidine deaminase (CDA) catalyzes hydrolyticdeamination of cytidine and deoxycytidine into uridine and deoxyuridine,respectively. Some known anticancer agents are nucleoside/nucleotideanalogs, for instance gemcitabine (2,2-difluorodeoxycytidine) andcytarabine (Ara-C, cytosine arabinoside). CDA problematically inactivatesuch anticancer agents, including gemcitabine and cytarabine. Asdemonstrated in the present Examples, the compounds of the inventionexert their cytotoxic effects independent of the expression of CDA.

Accordingly, in preferred embodiments, the cancer/tumour is one in whichCDA is expressed, preferably in which CDA is:

-   -   i) over-expressed;    -   ii) not over-expressed;    -   iii) under-expressed;    -   iv) not under-expressed;    -   v) over-expressed or under-expressed; or    -   vi) neither over-expressed nor under-expressed.

Each of cancer/tumour types i) to vi) above represents a preferredembodiment of the present invention. In a particularly preferredembodiment, the cancer/tumour treated according to the present inventionis one in which CDA is not over-expressed.

In the context of cancer/tumour cell gene expression, the terms“over-expressed” and “under-expressed” have a clear and widelyunderstood meaning, namely at increased/higher or decreased/lowerlevels, respectively.

Over-expression (or increased or higher levels) or under-expression (ordecreased or lower levels) may be as determined in comparison to anyappropriate control (e.g. control level or control sample or biopsy).For example, the control level may be the level in a sample (e.g. bloodor serum sample or tissue sample or biopsy) from a healthy subject (e.g.a subject not having cancer). Appropriate control levels (or controlsamples or values) could be readily chosen by a person skilled in theart. Appropriate control “values” could also be readily determinedwithout running a control “sample” in every test, e.g. by reference tothe range for normal subjects.

Preferably, the terms over-expressed and under-expressed mean that thelevel of RNA transcript from the gene in question in the cancerous cellsis higher (over-expressed) or lower (under-expressed) than the level innon-cancerous cells from the same tissue as the cancerous cells, asassessed using the same method and conditions in both cases. Preferredmethods and conditions for assessing the level of gene expression, e.g.CDA expression, are as disclosed elsewhere wherein.

Preferably, the over-expression or under-expression is significant. Bysignificantly overexpressed/under-expressed, i.e. significantlyhigher/lower is meant statistically significantly over/under expressed(statistically significantly higher/lower). By statistically significantis meant that the observed increased or decreased level is greater thanwhat might be expected to happen by chance alone. Statisticalsignificance is determined by any method known in the art. For examplestatistical significance is determined by probability value (p-value).The p-values is a measure of probability that a difference betweengroups during an experiment happened by chance. For example, a p-valueof 0.01 means that there is a 1 in 100 chance the result occurred bychance. The lower the p-value, the more likely it is that the differencebetween groups was caused by treatment. Preferably, the probabilityvalue is <0.05 or <0.01.

In some embodiments, increased levels (i.e. over-expression) may be anincrease of at least 5%, at least 10%, at least 20%, at least 30%, atleast 40%, at least 50% (e.g. as compared to a control level). In someembodiments, decreased levels (i.e. under-expression) may be an decreaseof at least 5%, at least 10%, at least 20%, at least 30%, at least 40%,at least 50% (e.g. as compared to a control level).

Preferably, the level of gene expression in a cell of interest isdetermined by reference to a database containing such information.Resources such as The Cancer Genome Atlas (TCGA,https://cancergenome.nih.gov/), the EMBL-EBI expression atlas(https://www.ebi.ac.uk/gxa/home), the human protein atlas(https://www.proteinatlas.org/), GENEVESTIGATOR®(https://genevestigator.com/gv/), the Cancer Cell Line Encyclopaedia(https://portals.broadinstitute.org/ccle) and others contain informationon gene expression levels in a wide range of cell types, includingcancers, as well as providing statistical data on whether or not thelevel of gene expression in a particular cancer type is significantlyhigher or lower than in non-cancerous cells from the same tissue (i.e.whether the gene is over-expressed or under-expressed in the cancertype). These databases are preferred. Thus, cancer types withstatistically significant over or under expression of a gene of interestcan be readily identified. It is within the abilities of the person ofordinary skill in the art to utilise such resources for this purpose.

Thus preferably, the cancer/tumour is as defined anywhere herein whereinthe CDA expression within said cancer/tumour is determined by referenceto a database selected from the EMBL-EBI expression atlas database(https://www.ebi.ac.uk/gxa/home), the GENEVESTIGATOR® database(https://genevestigator.com/gv/), the Cancer Cell Line Encyclopaedia(https://portals.broadinstitute.org/ccle) and the human protein atlas(https://www.proteinatlas.org/).

Preferably, the cancer/tumour does not express CDA to a higher levelthan the expression level in non-cancerous cells from the same tissue ascancer/tumour, wherein said over-expression is determined by referenceto a database selected from the EMBL-EBI expression atlas database(https://www.ebi.ac.uk/gxa/home), the GENEVESTIGATOR® database(https://genevestigator.com/gv/), the Cancer Cell Line Encyclopaedia(https://portals.broadinstitute.org/ccle) and the human protein atlas(https://www.proteinatlas.org/).

Preferably, the cancer/tumour is one wherein the CDA expression level isnot greater than 90% of the CDA expression level in a reference cancercell line as determined using the same method under the same conditions,wherein said reference cancer cell line is MDA-MB-231.

MDA-MB-231 is a well-characterised cell line which is widely availablecommercially. The MDA-MB-231 cell line is an epithelial, human breastcancer cell line that was established from a pleural effusion of a51-year-old caucasian female with a metastatic mammary adenocarcinomaand is one of the most commonly used breast cancer cell lines in medicalresearch laboratories. It can be obtained, for instance, from theEuropean Collection of Authenticated Cell Cultures (ECACC), catalogueno. 92020424. As shown in Table 4A, the expression level of CDA in theMDA-MB-231 cell line is 153 TPM.

MDA-MB-231 is a highly aggressive, invasive and poorly differentiatedtriple-negative breast cancer (TNBC) cell line as it lacks oestrogenreceptor (ER) and progesterone receptor (PR) expression, as well as HER2(human epidermal growth factor receptor 2) amplification. The cell lineis recognised as belonging to the claudin-low molecular subtype as itexhibits down-regulation of claudin-3 and claudinin-4, low expression ofthe Ki-67 proliferation marker, enrichment for markers associated withthe epithelial-mesenchymal transition and the expression of featuresassociated with mammary cancer stem cells (CSCs), such as theCD44+CD24−/low phenotype. In 3D culture, the cell line displaysendothelial-like morphology and is distinguished by its invasivephenotype, having stellate projections that often bridge multiple cellcolonies. Standard conditions for the culturing of this cell line arewell-known. Preferred culture conditions are growth at 37° C. inLeibovitz's L-15 medium supplemented with 2 mM glutamine and 15% foetalbovine serum (FBS). This medium supports the growth of cells inenvironments without CO2 equilibration. MDA-MB-231 cells are preferablyseeded at a density between 1-3×10⁴ cells/cm² and subcultured when70-80% confluent.

Preferably, the cancer/tumour is one wherein the CDA expression level isnot greater than 80%, preferably not greater than 70%, preferably notgreater than 60%, preferably not greater than 50%, preferably notgreater than 40%, preferably not greater than 30%, preferably notgreater 25% of the CDA expression level in a reference cancer cell lineas determined using the same method under the same conditions, whereinsaid reference cancer cell line is MDA-MB-231.

Preferably, the cancer/tumour is one wherein the CDA expression level isat least 2-fold lower, preferably at least 2.5-fold lower, preferably atleast 3-fold lower, preferably at least 3.5 fold lower, preferably atleast 4 fold lower, preferably at least 4.5 fold lower than the CDAexpression level in a reference cancer cell line as determined using thesame method under the same conditions, wherein said reference cancercell line is MDA-MB-231. “At least X-fold lower” in this context meansthat the maximum CDA expression level in the cancer/tumour is the valuethat is exactly X-fold lower than the expression level in the referencecancer cell line.

The method and conditions used to determine the CDA expression level inthe cancer/tumour and in the reference cancer cell line may be anysuitable method and conditions, provided that the same method andconditions are used to determine the level of CDA expression in thecancer/tumour as are used to determine the level of CDA expression inthe reference cancer cell line. The reference cancer cell line is thus acontrol. The person of ordinary skill in the art is readily able todetermine the expression level of a gene of interest, e.g. CDA, incancerous and non-cancerous cells alike. Such methods are part of thecommon general knowledge in the field and any suitable method may beused in the context of the present invention.

For instance, the expression level may be measured at the protein level.Methods for measuring protein expression levels are well-known in theart. Those methods generally involve contacting a biological sample ofinterest with one or more detectable reagents that is or are suitablefor measuring the protein's expression level, such as an antibody, andsubsequently determining the protein expression level based on the levelof detected reagent, preferably after normalization. Examples of methodswhich generally involve the use of an antibody include, withoutlimitation, Western blot, immunoblot, enzyme-linked immunosorbant assay(ELISA), enzyme-linked immunospot (ELISPOT), radioimmunoassay (RIA),immunohistochemistry and immunoprecipitation. Other methods suitable formeasuring a protein expression level, which do not necessarily involvethe use of an antibody, may be used, including, without limitation,fluorescence activated cell sorting (FACS), microscopy such as atomicforce microscopy, flow cytometry, microcytometry, protein binding assay,ligand binding assay, microarray, polyacrylamide gel electrophoresissuch as SDS-PAGE, surface plasmon resonance (SPR), Forster resonanceenergy transfer (FRET), Bioluminescence resonance energy transfer(BRET), chemiluminescence, fluorescent polarization, phosphorescence,mass spectrometry such as liquid chromatography mass spectrometry(LC-MS) or liquid chromatography/mass spectrometry/mass spectrometry(LC-MS-MS), matrix-assisted laser desorption/ionization time-of-flight(MALDI-TOF), surface-enhanced laser desorption/ionization time-of-flight(SELDI-TOF), and magnetic resonance imaging (MRI).

Preferably, however, the gene expression level, e.g. the CDA expressionlevel, may be measured at the RNA level. Methods for measuring RNAlevels are well-known in the art and any suitable method may be used inthe context of the present invention. For instance, microarray, RT-PCR,quantitative real time PCR, RNA sequencing, northern blots, primerextension, RNase protection and RNA expression profiling methods may beused. Preferably, the method used is RNA-seq or microarray. RNA-Seq is awell-known method, which uses next-generation sequencing (NGS) todetermine the presence and quantity of RNA in a biological sample at agiven moment

Preferably, the method used to determine the CDA expression level in acancer/tumour of interest as compared to the CDA expression level in areference cancer cell line, wherein said reference cancer cell line isMDA-MB-231, is “Microarray Method A” referred to below.

Microarray Method A comprises the following steps:

-   1) Extracting RNA from cancer/tumour cells of interest, said    extraction preferably being performed using a Norgen Total RNA    Purification kit (Norgen Biotek Cat nr. 17200);-   2) Preparing Poly(A)+RNA from the extracted RNA, preferably using a    MEGApure kit according to the manufacturer's instructions (Ambion);-   3) Preparing complementary DNA (cDNA) from the Poly(A)+RNA by    treatment with reverse transcriptase, i.e. performing reverse    transcription;-   4) Fragmenting the cDNA using TdT (terminal deoxynucleotidyl    transferase);-   5) Biotinylating the cDNA fragments using the GeneChip WT Terminal    labelling kit (Affymetrix);-   6) Repeating steps 1 to 5 with a reference cell line, wherein said    reference cell line is MDA-MB-231;-   7) Hybridizing 5.5 μg of the biotinylated cDNA fragments obtained in    step 5 to a first DNA microarray at 45° C. for 16 hours, and    hybridizing 5.5 μg of the biotinylated cDNA fragments obtained in    step 6, to a DNA microarray at 45° C. for 16 hours, preferably    wherein said microarrays are Affymetrix GeneChip Human Gene 2.0 ST    Arrays (Applied Biosystems);-   8) Washing and staining the hybridized microarrays, preferably in    the Affymetrix GeneChip Fluidics Station 450 (Applied Biosystems);-   9) Scanning said stained microarrays with the Affymetrix GeneChip    Scanner 3000 7G utilising the Affymetrix GeneChip Command Console®    software to produce raw data signal values in the form of CEL files;-   10) Normalizing the CEL files to produce gene-level expression    values using the implementation of the Robust Multiarray Average    (RMA) in the Affymetrix software package (version 1.36.1),    preferably as described in R. A. Irizarry et al., Exploration,    normalization, and summaries of high density oligonucleotide array    probe level data. Biostatistics 4, 249-264 (2003);-   11) Assessing array quality by computing relative log expression    (RLE) and normalized unscaled standard error (NUSE) using the    affyPLM package (version 1.34.0). The output from the affyPLM    package is boxplots for both the RLE and NUSE distributions. Arrays    for which the RLE boxplots are not centered around 0, and arrays for    which the NUSE boxplots are not centered around 1, are flagged as    poor quality and are excluded from subsequent analysis. If the array    is excluded, then the previous steps of the method are repeated.-   12) Performing Principal Component Analysis (PCA) using the Prcomp R    function with expression values that have been normalized across all    samples to a mean of zero and a standard deviation of one. PCA is    used as a dimensionality reduction method in order to reduce the    high dimensionality of the gene expression dataset while retaining    most of the variation in the data. Thus, the performance of PCA    achieves a lower dimensional representation of the data for    downstream analysis and visualization.-   13) Assessing differential expression of CDA in the cancer/tumour    cells of interest and in the reference cell line MDA-MB-231 using    the moderated (empirical Bayesian) t-test implemented in the limma    package (version 3.14.4, http//bioinf.wehi.edu.au/imma);    wherein the microarray analysis steps 10 to 13 are performed using    the R environment for statistical computing (version 2.15.1).

Preferably, the CDA expression level in a cancer/tumour of interest ascompared to the CDA expression level in a reference cancer cell line,wherein said reference cancer cell line is MDA-MB-231, is determined byreference to a database selected from the EMBL-EBI expression atlasdatabase (https://www.ebi.ac.uk/gxa/home), the GENEVESTIGATOR® database(https://genevestigator.com/gv/), the Cancer Cell Line Encyclopaedia(https://portals.broadinstitute.org/ccle) and the human protein atlas(https://www.proteinatlas.org/).

The expression level of a gene is typically presented in terms of therelative amount of RNA transcript for that gene as compared to the totalamount of RNA transcripts in the cell/tissue concerned. The expressionlevel is typically presented in units of transcripts per million (TPM),that is for every 1 million RNA molecules within the cell/tissue ofinterest, [x] many came from the gene of interest. Again, the level ofRNA transcript in terms of TPM can be obtained by the skilled person byroutine methods such as quantitative real time PCR or RNA sequencingmethods, and such information is available from resources such as TCGA,the EMBL-EBI expression atlas, the GENEVESTIGATOR® database(https://genevestigator.com/gv/), the Cancer Cell Line Encyclopaedia(https://portals.broadinstitute.org/ccle) and the human protein atlas(https://www.proteinatlas.org/), amongst others. Such methods arepreferred herein. Methodology for the determination of gene expressionlevels in TPM are described in the literature, for instance, in Wagneret al., (2012) Theory Biosci 131(4):281-285 or Mortazavi A et al.,(2008) “Mapping and quantifying mammalian transcriptomes by RNA-Seq.”Nature methods 5(7):621-8.

Preferably, cancerous cells/tumours which over-express CDA contain CDARNA transcripts at a level greater than 140 TPM. Conversely, cancerouscells/tumours which do not over-express CDA preferably contain CDA RNAtranscripts at a level ≤140 TPM (less than or equal to 140 TPM).Alternatively viewed, the preferred cancerous cells/tumours of theinvention have a CDA expression level of ≤140 TPM. Thus, particularlypreferred cancers/tumours to be treated in accordance with the presentinvention are those which express CDA to a level of ≤140 TPM, morepreferably ≤100 TPM, more preferably ≤50 TPM.

Preferably, the expression level of CDA in a cancer/tumour of interest,in units of TPM, is obtained by reference to a database selected fromthe EMBL-EBI expression atlas database (https://www.ebi.ac.uk/gxa/home),the GENEVESTIGATOR® database (https://genevestigator.com/gv/), theCancer Cell Line Encyclopaedia (https://portals.broadinstitute.org/ccle)and the human protein atlas (https://www.proteinatlas.org/).

Preferably, the level of CDA in units of TPM in a cancer/tumour ofinterest is determined by quantitative real time PCR, or RNA sequencing(RNA-seq) and quantification, using cells derived from saidcancer/tumour. Preferably, the method used is RNA sequencing.Preferably, the method used is ““RNA-seq Method A” referred to below.

The expression level of a gene in units of TPM can be determined usingany known method but is preferably determined using RNA-seq. RNA-seqmethods are well-known in the art and are widely available commercially.Any suitable RNA-seq method can be used in order to determine the CDAexpression level in units of TPM in a cancer of interest. The followingmethod, termed RNA-seq Method A, is preferred, and comprises thefollowing steps:

-   -   1. Extracting total RNA from the cancer/tumour cells of        interest. This may be performed using standard RNA extraction        kits, preferably QIAGEN AllPrep DNA/RNA Mini kit (Hilden,        Germany) according to the manufacturer's instructions.    -   2. Optionally quantifying and assessing the extracted RNA for        purity, preferably by an automated electrophoresis tool,        preferably a 2100 Bioanalyzer (Agilent Technologies). The        Agilent Bioanalyzer is a microfluidics platform used for sizing,        quantification, and quality control for RNA (and DNA/proteins)        and provides an “RNA Integrity Number” (RIN), which quantifies        the fragmentation of the RNA sample. Preferably, samples are        only used further in the process if they have an RIN of at least        7, preferably at least 8.    -   3. Preparing an RNA-sequencing library, preferably using the        Illumina TruSeq™ RNA Sample Preparation Kit (Illumina, San        Diego, Calif., USA) and the associated protocol. Preferably        500-1000 μg of total RNA is used per sample in this process.        This kit and protocol is well-known in the field. This step        comprises the following steps:        -   3a. Treating the extracted RNA to deplete bacterial and            eukaryotic ribosomal RNA, preferably using the Ribo-Zero            rRNA Removal Kit (Epicentre, Madison, Wis., USA), following            the manufacturer's instructions        -   3b. Treating the remaining RNA with reverse transcriptase            and random hexamers to create single-stranded cDNA fragments            of 100-150 bases in length, or preferably 200-300 bases in            length.        -   3c. Treating the single-stranded cDNA fragments with DNA            Polymerase I and RNase H to produce double-stranded            complemented DNA fragments (cDNA); and        -   3d. Ligating RNA-seq adaptors to the termini of the cDNA            fragments to produce a library of adaptor-cDNA sequences            capable of being analysed by RNA-seq. RNA-seq adapters are            well known in the field and any suitable adapters may be            used. Adapters contain functional elements which permit            sequencing; for example, an amplification element and a            primary sequencing site. Preferably, the adaptors are            Illumina indexing adaptors. Adaptors may be ligated to one            end of each cDNA fragment to produce single-end libraries            (which will result in one “read” per fragment when            sequenced), in which case the cDNA fragment length in step            3b is 100-150 bases. Preferably however, adaptors are            ligated to both ends of each cDNA fragment to produce            paired-end libraries (which will result in two “reads” per            fragment when sequenced, so-called “mate reads” or “paired            reads”), in which case the cDNA fragment length in step 3b            is 200-300 bases.    -   4. Optionally amplifying the adaptor-cDNA sequences by PCR. This        step may be performed if the amount of DNA is insufficient for        sequence analysis step 5 to be performed. The amount of DNA        required for sequence analysis is well-known to the skilled        person (preferably 70-135 μl, more preferably 125 μl, of a 20 pM        solution of DNA is loaded into the flow cell of the sequencer in        step 5). Methods of assessing the amounts of DNA in a sample are        routine, and any suitable method may be used. Preferably, the        method used is fluorometry, preferably a Qubit® fluorometer is        used.    -   5. Sequencing the adaptor-cDNA sequences, preferably using an        Illumina Flowcell sequencer, preferably a Illumina Flowcell        HiSeq 2500 sequencer or an Illumina Flowcell NovaSeq 6000        sequencer, preferably using a sequencing cycle of 100-150 base        pairs if single-end libraries are produced in step 3, more        preferably using a sequencing cycle of 200-300 base pairs with        paired-end libraries being produced in step 3. Preferably 70-135        μl, more preferably 125 μl, of a 20 pM solution of DNA is loaded        into the flow cell. The raw data set obtained will provide a        unique sequence identifier for each fragment analysed, its        sequence (a so-called “read”), and an indication of the        confidence in the sequencer's determination of the sequence at        each base position within the read (a so-called Phred quality        score).    -   6. Preparing a quality-filtered data set of reads by removing        from the data set of determined sequences:        -   i) those bases within a read which have a Phred quality            score of less than 10;        -   ii) those bases within a read which are downstream of (i.e.            subsequent in the direction of sequencing to) a base in the            same read having a Phred quality score of less than 10;        -   iii) those reads which contain any undetermined (“uncalled”)            bases (“N”);        -   iv) those reads which map to a contaminant reference genome,            wherein said contaminant reference genome is the E. coli            genome; and        -   v) if the RNA-seq library used is a paired-end library,            those reads whose “mate read” was discarded in one of steps            6(iii) to 6(iv).    -   7. Aligning the quality-filtered reads to the genome of the        subject species, i.e. preferably the human genome. Preferably        quality-filtered reads are aligned to the human reference genome        from the Ensembl database version 98, preferably genome Human        GRCh38 and Ensembl gene reference feature database (version        Ensembl Genomes 45, GENCODE 32). Alignment tools suitable for        this step are well-known, widely available and any suitable        alignment tool can be used. Preferably, the alignment tool is        TopHat2 (version 2.1.1) [Trapnell et al., (2010) Nature        Biotechnology 28, 511-515].    -   8. Determining the number of read counts for the CDA gene, and        optionally any other genes of interest. Bioinformatic tools for        this step are well-known, widely available and any suitable tool        can be used. Preferably, read counts per gene are determined        using HTSeq package (htseq-count). This step thereby provides        raw read count data.    -   9. Converting the raw read count data obtained in step 8 into        units of transcripts per million (TPM). This is a standard        mathematical operation, routinely used in the field. Providing        count data in units of TPM allows for a meaningful assessment of        gene expression levels because the determination of TPM values        involves normalizing the raw data for i) Gene length; and ii)        Sequencing depth.        -   i) Regarding gene length normalization, in the raw data, a            higher read count may be observed for longer genes purely            because the genes are longer and so more fragments align            with that gene. Normalizing for gene length comprises            dividing the read counts for each gene by the gene length            (in kilobases).        -   ii) Sequencing depth is a between-sample effect that alters            the comparison of read counts between the same gene in            different samples. In order to normalize this, the read            counts per kilobase obtained in step 9i) are divided by a            “per million scaling factor”, which is itself obtained by            dividing the total number of reads in a sample by 1 million.

Gene expression data obtained using RNA-seq can be presented in units ofRPKM (Reads Per Kilobase Million). This is calculated by

-   -   i. Dividing the total number of reads in a sample by 1,000,000        to provide a “per million scaling factor”;    -   ii. Dividing the read counts per gene by the “per million”        scaling factor, which normalizes for sequencing depth, thereby        providing units of reads per million (RPM); and    -   iii. Dividing the RPM values by the length of the gene, in        kilobases, which normalizes for gene length, thereby providing        units of RPKM.

Another unit of gene expression is Fragments Per Kilobase Million(FPKM), which is very similar to RPKM. RPKM is applicable whensingle-end RNA-seq is used, where every read corresponds to a singlefragment that was sequenced. FPKM is applicable for paired-end RNA-seq,wherein two reads can correspond to a single fragment, or, if one readin the pair did not map, one read can correspond to a single fragment.The only difference between RPKM and FPKM is that FPKM takes intoaccount that two reads can map to one fragment (and so it doesn't countthis fragment twice).

TPM is very similar to RPKM and FPKM. The only difference is the orderof operations (i) to (iii) above. Thus, TPM is determined as follows:

-   -   i. Dividing the read counts per gene by the length of the gene,        in kilobases, which normalizes for gene length, thereby        providing units of reads per kilobase (RPK);    -   ii. Dividing the total RPK value in a sample by 1,000,000 to        provide a “per million scaling factor”; and    -   iii. Dividing the RPK values obtained in step (i) by the “per        million” scaling factor obtained in step (ii), which normalizes        for sequencing depth, thereby providing units of TPM.

The only difference when calculating TPM as compared to RPKM or FPKM isthat when calculating TPM normalization for gene length is performedfirst. However, the result is that when using units of TPM, the sum ofall TPMs in each sample are the same—1 million. This allows moremeaningful comparison of the proportion of reads that mapped to a genein each sample.

The above described RNA-seq Method A, of determining the expressionlevel of CDA in units of TPM can also be used to determine theexpression level of CDA in a cancer/tumour of interest, in units of TPM,as compared to a control, i.e. control cells, as defined above or areference cell line, wherein said reference cell line is MDA-MB-231. Inthese embodiments, the RNA-seq Method A may be performed using thecancer/tumour cells of interest and repeated with the control orreference cells, wherein the TPM values determined are then compared.Alternatively, the RNA-seq Method A may be performed using thecancer/tumour cells of interest and the CDA expression level determinedin units of TPM is compared to a value for the CDA expression level inunits of TPM previously obtained using the control or reference cellline using the same method.

Alternatively, the level of expression of a gene can be determined viamicroarray, which is a standard technique in the field. Any suitablemicroarray technique can be used in the context of the presentinvention. Microarrays permit the detection of expression of thousandsof genes simultaneously.

The level of gene expression determined by microarray can be expressedon any scale, e.g. a linear scale. Data from microarrays may also betransformed, preferably by the logarithm base 2 transformation, whichhas the advantage of producing a continuous spectrum of values andtreating up- and downregulated genes in a similar fashion. A geneup-regulated by a factor of 2 has a log 2 transformed value of 1.

Preferably, the cancer/tumour is one in which the logarithm base 2transformed CDA expression level is less than 11.75. Suchcancers/tumours are described as not over-expressing CDA. Preferably thecancer/tumour is one in which the logarithm base 2 transformed CDAexpression level is less than 11.5, more preferably less than 11, morepreferably less than 10.5, more preferably less than 10, more preferablyless than 9.5.

Preferably, the cancer/tumour is one in which the linear CDA expressionlevel is less than 6500. Such cancers/tumours are described as notover-expressing CDA. Preferably the cancer/tumour is one in which thelinear CDA expression level is less than 6000, more preferably less than5000, more preferably less than 4000, more preferably less than 3000,more preferably less than 2000, more preferably less than 1500.

Preferably, the linear or logarithm base2 transformed expression levelof CDA in a cancer/tumour of interest is obtained by reference to adatabase selected from the EMBL-EBI expression atlas database(https://www.ebi.ac.uk/gxa/home), the GENEVESTIGATOR® database(https://genevestigator.com/gv/), the Cancer Cell Line Encyclopaedia(https://portals.broadinstitute.org/ccle) and the human protein atlas(https://www.proteinatlas.org/).

The skilled person is aware that there may be variation in expressionlevels of some genes between tumours of the same cancer type. Forinstance, some breast cancers may over-express CDA and others may notover-express CDA. Thus, preferably, the cancers/tumours describedanywhere elsewhere herein as preferred are preferably cancers/tumours ofthose types in which CDA is expressed, preferably in which CDA is:

-   -   i) over-expressed;    -   ii) not over-expressed;    -   iii) under-expressed;    -   iv) not under-expressed;    -   v) over-expressed or under-expressed; or    -   vi) neither over-expressed nor under-expressed.        Preferably, the cancer/tumour is one in which CDA is not        over-expressed. Preferably, the cancer/tumour is one in which        CDA is over-expressed. CDA over-expression is as defined above.

Conversely, the cancers described anywhere else herein as not preferredare preferably only cancers/tumours of those types in which CDA is:

-   -   i) over-expressed;    -   ii) not over-expressed;    -   iii) under-expressed;    -   iv) not under-expressed;    -   v) over-expressed or under-expressed; or    -   vi) neither over-expressed nor under-expressed.        Preferably, the cancer/tumour is not one in which CDA is        over-expressed. CDA over-expression is as defined above.

In a preferred embodiment, the cancer/tumour is resistant to gemcitabineand/or cytarabine treatment. Thus, the cancers/tumours describedanywhere elsewhere herein as preferred are preferably cancers/tumours ofthose types which are resistant to gemcitabine and/or cytarabinetreatment treatment. Preferably the gemcitabine and/or cytarabineresistant cancer/tumour is brain cancer, preferably glioma, morepreferably glioblastoma multiforme.

The human protein 0-6-methylguanine-DNA methyltransferase (MGMT) removesalkylated DNA damage. The expression of MGMT makes cancer cells, such asglioblastoma cells, almost completely resistant to the cytotoxic effectsof Temozolomide, which exerts its cancer chemotherapeutic activity bymutating

tumor cells so severely that the tumor cells are killed. Temozolomideworks by alkalyting DNA, thereby causing mutations.

As shown in the present Examples, the compounds of Formula (I) are notmutagenic in a HPRT assay. Accordingly, the compounds of Formula (I) areof use particular use in treating cancers in which MGMT is expressed,preferably over-expressed. Thus, the cancers/tumours described anywhereelsewhere herein as preferred are preferably cancers/tumours of thosetypes in which MGMT is expressed, preferably over-expressed.

Temozolomide(4-methyl-5-oxo-2,3,4,6,8-pentazabicyclo[4.3.0]nona-2,7,9-triene-9-carboxamide)is the first-line treatment for glioblastoma multiforme, and is alsoused in the treatment of some other brain cancers. As shown in thepresent Examples however, temozolomide effectively kills less than halfof glioblastoma cell lines evaluated, and temozolomide resistant celllines are effectively killed by the compounds of the invention. Thus, ina preferred embodiment, the cancer/tumour is temozolomide resistantcancer/tumour.

Thus, the cancers/tumours described anywhere elsewhere herein aspreferred are preferably cancers/tumours of those types which areresistant to Temozolomide treatment. Preferably, the temozolomideresistant cancer/tumour is brain cancer, preferably glioma, morepreferably glioblastoma multiforme.

As shown in the present Examples, 5-fluorouracil resistant cell linesare effectively killed by the compounds of the invention. Thus, in apreferred embodiment, the cancer is fluoropyrimidine resistant cancer.Thus, the cancers/tumours described anywhere elsewhere herein aspreferred are preferably cancers/tumours of those types which areresistant to fluoropyrimidine treatment. preferably the fluoropyrimidineresistant cancer/tumour is brain cancer, preferably glioma, morepreferably glioblastoma multiforme. Preferably the fluoropyrimidine is5-fluorouracil.

Preferably the cancer/tumour is resistant to both Temozolomide andfluoropyrimidine treatment.

The term “resistant” in the context of cancer/tumour therapy has a clearand well-understood meaning in the art. By “resistant” is meant that thecancer/tumour does not respond positively to treatment with theanticancer agent(s) concerned, i.e. that treatment with the anticanceragent(s) does not reduce, alleviate, ameliorate or eliminate the cancer,or one or more symptoms thereof, or reduce or eliminate cancer cellswithin the tumour, relative to the cancer, tumour or symptom prior tothe treatment. A cancer/tumour may be resistant at the beginning oftreatment, or it may become resistant during treatment.

As mentioned above, in the treatment or prevention of cancer thecompound of Formula (I) may be used alone or optionally in combinationwith a further, i.e. one or more further, anticancer agent(s). In allaspects and embodiments of the present invention, the further anticanceragent may be any suitable anti-cancer agent known in the art. A widerange of different types of agents are known or proposed for use in thetreatment of cancer and any of these may be used, regardless of chemicalnature or mode of action.

Anticancer agents thus included chemical molecules whether naturally orsynthetically derived or prepared (e.g organic small chemical molecules)and biological molecules such as proteins and peptides (e.g.immunotherapy agents as discussed below). Anticancer drugs thus includechemotherapeutic agents or drugs, which may be in a wide range ofdifferent chemical or functional classes, as well as antibodies orantibody derivatives and other biological molecules which act forexample to stimulate, activate or enhance various physiologicalprocesses or cells in the body, for example immune and/oranti-inflammatory responses or cells etc.

Representative examples of anticancer agents in the “chemotherapy” classinclude but are not limited to fludarabine, gemcitabine, capecitabine,methotrexate, taxol, taxotere, mercaptopurine, thioguanine, hydroxyurea,cytarabine, cyclophosphamide, ifosfamide, nitrosoureas, platinumcomplexes such as cisplatin, carboplatin and oxaliplatin, mitomycin,dacarbazine, procarbazine, etoposide, teniposide, campathecins,bleomycin, doxorubicin, idarubicin, daunorubicin, dactinomycin,plicamycin, mitoxantrone, L-asparaginase, epimbicm, 5-fluorouracil,taxanes such as docetaxel and paclitaxel, leucovorin, levamisole,irinotecan, estramustine, etoposide, nitrogen mustards, BCNU,nitrosoureas such as carmustme and lomustine, vinca alkaloids such asvinblastine, vincristine and vinorelbine, imatimb mesylate,hexamethyhnelamine, or topoteca.

Anticancer agents may include kinase inhibitors, phosphatase inhibitors,ATPase inhibitors, tyrphostins, protease inhibitors, herbimycin A,genistein, erbstatin, and lavendustin A.

In one embodiment, the anticancer agent may be selected from, but is notlimited to, one or a combination of the following class of agents:alkylating agents, plant alkaloids, DNA topoisomerase inhibitors,anti-folates, pyrimidine analogs, purine analogs, DNA antimetabolites,taxanes, podophyllotoxin, hormonal therapies, retinoids,photosensitizers or agents for use in photodynamic therapies,angiogenesis inhibitors, antimitotic agents, isoprenylation inhibitors,cell cycle inhibitors, actinomycins, bleomycins, anthracyclines, MDRinhibitors and Ca2+ ATPase inhibitors.

Further anticancer agents may be selected from, but are not limited to,cytokines, chemokines, growth factors, growth inhibitory factors,hormones, soluble receptors, decoy receptors, monoclonal or polyclonalantibodies, mono-specific, bi-specific or multi-specific antibodies,monobodies, polybodies.

Alternative anticancer agents may be selected from, but are not limitedto, growth or hematopoietic factors such as erythropoietin andthrombopoietin, and growth factor mimetics thereof.

In one representative embodiment the drug is a small molecule, and moreparticularly a small molecule chemotherapeutic agent. A small moleculeagent may be defined as having a molecular weight of less than 2000 Da,more particularly less than 1800, 1500, 1200, 1000, 900, 800 or 700 Da.,typically less than 1000 Da. For example, a small molecule agent mayhave a size in the range of 100-1000 Da, e.g. 100-800 Da or 300-700 Da.

In an alternative embodiment, the further anticancer agent is animmunotherapy agent. Induction of an immune response to treat cancer isknown as cancer “immunotherapy”. Immunotherapy can involve, for example,cell-based therapies, antibody therapies or cytokine therapies. Allthree approaches exploit the fact that cancer cells often have differentcell-surface markers, or cancer antigens, which are detectable by theimmune system. These antigens are most commonly proteins but may alsoinclude other molecules such as carbohydrates. Another example ofimmunotherapy is by checkpoint inhibition, whereby checkpoint proteinsare inhibited. This is discussed further below.

Immunotherapy is thus used to provoke the immune system into attackingcancer cells, and as discussed further below, various molecules may bethe target of immunotherapy-based approaches. For example, targets forimmunotherapeutic intervention in cancer include “CD” (“cluster ofdifferentiation”) proteins such as CD52, CD30, CD33, CD20, CD152 (alsoknown as CTLA4) and CD279 (also known as programmed cell death 1 proteinPD-1); growth factors such as vascular endothelial growth factor (VEGF);growth factor receptors such as epidermal growth factor receptor (EGFR)or human epidermal growth factor receptor 2 (HER2);Lymphocyte-activation gene 3 (LAG3); and B7 family proteins such asB7-H3 and B7-H4. These are merely representative examples however, andother molecules may be targets for immunotherapeutic intervention incancer.

The immunotherapy agent can be a peptide, polypeptide or protein. Theimmunotherapy agent may be selected from an antibody, a cytokine and acheckpoint inhibitor. As noted above a therapeutic anticancer antibodymay have a range of targets, including checkpoint proteins. Thus, anantibody may be a checkpoint inhibitor.

Thus, in a first example, the immunotherapeutic agent is an antibody.The antibody may be selected from monoclonal or polyclonal antibodies,mono-specific, bi-specific or multi-specific antibodies, monobodies andpolybodies or indeed from any of the many antibody-like or antibodyderivative molecules known in the art today. Accordingly the term“antibody” is used broadly herein and includes any such antibody and anyantibody fragment, derivative or variant as in the known in the art. Theantibody may be of any convenient or desired species, class or sub-type.Furthermore, the antibody may be natural, derivatised or synthetic.

The antibody may accordingly be:

-   -   (a) any of the various classes or subclasses of immunoglobulin        e.g. IgG, IgA, IgM, IgD or IgE derived from any animal e.g. any        of the animals conventionally used e.g. sheep, rabbits, goats,        or mice or egg yolk;    -   (b) monoclonal or polyclonal antibodies;    -   (c) intact antibodies or fragments of antibodies, monoclonal or        polyclonal, the fragments being those which contain the binding        region of the antibody e.g. fragments devoid of the Fc portion        (e.g. Fab, Fab′, F(ab′)2, Fv), the so called “half molecule”        fragments obtained by reductive cleavage of the disulphide bonds        connecting the heavy chain components in the intact antibody. Fv        may be defined as a fragment containing the variable region of        the light chain and the variable region of the heavy chain        expressed as two chains;    -   (d) antibodies produced or modified by recombinant DNA or other        synthetic techniques, including monoclonal antibodies, fragments        of antibodies, humanised antibodies, chimeric antibodies, or        synthetically made or altered antibody-like structures.

Also included are functional derivatives or “equivalents” of antibodiese.g. single chain antibodies. A single chain antibody may be defined asa genetically engineered molecule containing the variable region of thelight chain, the variable region of the heavy chain, linked by asuitable polypeptide linker as a fused single chain molecule. Methodsfor producing antibodies and the fragments and derivatives of theantibodies are well known in the art.

In a preferred embodiment the antibody is a monoclonal antibody.

In many cases monoclonal antibodies are antibodies without modification,and most of the currently-used therapeutic antibodies fall into thiscategory. However, in one embodiment of the present invention theantibody, for example a monoclonal antibody, is conjugated or fused to afurther additional molecule, for example a toxic substance or aradioactive substance. Thus, conjugated or fused antibodies are joinedto another molecule, which is either toxic to cells (e.g. a drug) orradioactive. The antibody binds to specific antigens on the surface ofcancer cells and directs the toxin or radiation to the tumour.

Known and approved antibodies include: Alemtuzumab, Bevacizumab,Brentuximab vedotin, Cetuximab, Gemtuzumab ozogamicin, Ibritumomabtiuxetan, Ipilimumab, Ofatumumab, Panitumumab, Rituximab, Tositumomaband Trastuzumab.

In a second example, the immunotherapeutic agent is a cytokine.Cytokines include immunomodulating agents, such as interleukins (IL) andinterferons (IFN) and also colony stimulating factors, tumour necrosisfactors (TNF) and other regulatory molecules. Cytokines have beenclassed as lymphokines, interleukins, and chemokines, based on theirfunction, cell of secretion, or target of action. Each cytokine has amatching cell-surface receptor, which initiates cascades ofintracellular signalling which alter cell functions. In the context ofcancer, cytokines are produced by many cell types present within atumour. Cytokines are well known in the art and all such cytokines areencompassed for use according to the invention. As such, in oneembodiment the immunotherapeutic agent is a cytokine. In a preferredembodiment the cytokine is an interleukin or an interferon.

Interleukins are a group of cytokines with a wide array of effects onthe immune system. Examples of interleukins (ILs) are IL-1, IL-2, IL-3,IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-15and IL-17.

Interferons are cytokines produced by the immune system usually involvedin anti-viral response, but also have use in the treatment of cancer.There are three groups of interferons (IFNs): type I (IFNα and IFNβ),type 2 (IFNγ) and the relatively newly discovered type III (IFNA).

All known forms of the above-discussed cytokines can be used in thepresent invention, including also functionally equivalent variants,derivatives and fragments thereof. Thus the term “cytokine” as usedherein includes amino acid sequence variants of known cytokinepolypeptides, and fragments of a cytokine polypeptide, or derivativethereof, as long as such fragments, variants or derivatives are active,or “functional”, i.e. retain at least one function or activity (e.g.biological activity) of the relevant cytokine. The cytokine may be arecombinant polypeptide, a synthetic polypeptide or may be isolated froma natural source. Suitable cytokines are commercially available andwould be known to the skilled man, for example human cytokines areavailable from GenScript (Piscataway, N.J., USA).

In a third example, the immunotherapeutic agent is an agent that targetsan immune checkpoint, i.e. is a checkpoint inhibitor. Checkpointproteins keep the immune system in check by indicating to the immunesystem which cells are healthy and which cells should be destroyed.Checkpoint proteins act as a “brake” on the immune system by preventingT-cell activation. If a cell does not have sufficient checkpointproteins on its surface it may be destroyed by the immune system. In thecase of cancer cells, whilst there may be molecules signalling that thecell is cancerous, if there are enough checkpoint proteins on the cellsurface, the cell may evade the immune response, and it has beenspeculated that checkpoint proteins contribute to a lack of success insome cancer immunotherapies.

Several checkpoint inhibitors are known and can be used in the presentinvention, for example those inhibitors described in Creelan (2014)Cancer Control 21:80-89.

Examples of checkpoint inhibitors include: Tremelimumab (CP-675,206);Ipilimumab (MDX-010); Nivolumab (BMS-936558); MK-3475 (formerlylambrolizumab); Urelumab (BMS-663513); anti-LAG-3 monoclonal antibody(BMS-986016); and Bavituximab (chimeric 3G4). All of these checkpointinhibitors can be used in the present invention.

An alternative option for immunotherapy relates to immune celltherapies, and the present invention can also be used in combinationwith such therapies, for example adoptive cell transfer. A number ofT-cell-based therapies for treating cancer have been developed, andthese treatments, known as adoptive cell transfer (ACT) have becomeincreasingly attractive during recent years. Three main ACT strategieshave been exploited thus far. The first of these, and the mostdeveloped, involves the isolation of patient's own tumour-reactiveT-cells from peripheral or tumour sites (known as Tumour InfiltratingLymphocytes (TILs)). These cells are expanded ex vivo and re-injectedinto a patient.

Two alternative therapies are available, which involve modification of apatient's own T-cells with receptors capable of recognising a tumour. Inone option, TcRs having activity towards a cancer antigen can beisolated and characterised, and a gene encoding the TcR can be insertedinto T-cells and re-injected into a patient. This therapy has been shownto shrink solid tumours in some patients, but is associated with asignificant drawback: the TcRs used must be matched to a patient'simmune type. Accordingly, as an alternative to the use of TcRs,therapies involving the expression of Chimeric Antigen Receptors (CARs)in T-cells have also been suggested. CARs are fusion proteins comprisingan antibody linked to the signalling domain of the TcR complex, and canbe used to direct T cells against a tumour if a suitable antibody isselected. Unlike a TCR, a CAR does not need to MHC-matched to therecipient.

Alternatively, the cell may be a natural killer (NK) cell, whichoptionally may be modified to express a CAR.

As such, according to the present invention the immunotherapeutic agentmay be a cell, particularly an immune cell such as a lymphocyte,particularly a T cell or NK cell as described above, e.g. the T cell maybe a TIL or be modified to express a TcR or CAR. The NK may be modifiedto express a CAR.

An alternative option for the further anticancer agent is a microRNA(miRNA). MicroRNAs are small non-coding RNA molecule (containing about22 nucleotides) found in plants, animals, and some viruses, whichfunctions in RNA silencing and post-transcriptional regulation of geneexpression. miRNAs function via base-pairing with complementarysequences within mRNA molecules. As a result, these mRNA molecules aresilenced by cleavage of the mRNA strand into two pieces, destabilizationof the mRNA through shortening of its poly(A) tail, or less efficienttranslation of the mRNA into proteins. miRNAs resemble the siRNAsmentioned above, except miRNAs derive from regions of RNA transcriptsthat fold back on themselves to form short hairpins, whereas siRNAsderive from longer regions of double-stranded RNA. Many miRNAs have beenfound to have links with various types of cancer and accordingly aresometimes referred to as “oncomirs”.

MicroRNAs can be used in microRNA-based oncology therapeutics in thetreatment of cancer. The rationale for developing miRNA therapeutics isbased on the premise that aberrantly expressed miRNAs play key roles inthe development of cancers, and that correcting these miRNA deficienciesby either antagonizing or restoring miRNA function may provide atherapeutic benefit, e.g. by miRNA replacement therapy

Any suitable miRNA may be used as the further anticancer agent accordingto the present invention. The miRNA may be in free form, i.e. not boundto another molecule. Alternatively, the miRNA may be conjugated or boundto another molecule, e.g. an antibody as discussed herein.

Most preferably, the further anticancer agent is selected from the groupconsisting of temozolomide, 5-fluorouracil, gemcitabine, cytarabine andGliadel®. Preferably, the further anticancer agent is temozolomide.Preferably, the further anticancer agent is 5-fluorouracil. Preferablythe further anticancer agent is gemcitabine. Preferably the furtheranticancer agent is cytarabine. Preferably the further anticancer agentis Gliadel®.

The compound of Formula (I) and the further anticancer agent may be usedaccording to the present invention in the form of a composition, i.e. apharmaceutical composition. The present invention provides apharmaceutical composition comprising a compound of Formula (I) and oneor more pharmaceutically acceptable excipients, optionally furthercomprising a further anticancer agent.

The product (particularly a pharmaceutical product) comprising acompound of Formula (I) and a further (i.e. one or more further orsecond) anticancer agent(s) may be a combined preparation for separate,sequential or simultaneous use in the treatment or prevention of cancer.(i.e. of a tumour), or may be a product in which the compound of Formula(I) is co-formulated with a further anticancer agent.

Thus, the compound of Formula (I) and the further anticancer agent maybe formulated together in a single composition or in separatecompositions for separate administration. This will depend on the natureof the further anticancer agent and its selected or required mode ofadministration.

The compositions for use in the invention may be formulated in anyconvenient manner according to techniques and procedures known in thepharmaceutical art, e.g. using one or more pharmaceutically acceptablediluents, carriers or excipients. Such formulations may be forpharmaceutical or veterinary use. Suitable diluents, excipients andcarriers for use in such formulations are known to the skilled person.

“Pharmaceutically acceptable” as referred to herein refers toingredients that are compatible with other ingredients of thecompositions as well as physiologically acceptable to the recipient. Thenature of the composition and carriers or excipient materials, dosagesetc. may be selected in routine manner according to choice and thedesired route of administration, purpose of treatment etc.

Thus, “pharmaceutically” or “pharmaceutically acceptable” refers tomolecular entities and compositions that do not produce an adverse,allergic or other untoward reaction when administered to a mammal,especially a human, as appropriate. A pharmaceutically acceptablecarrier or excipient refers to a non-toxic solid, semi-solid or liquidfiller, diluent, encapsulating material or formulation auxiliary of anytype.

The pharmaceutical compositions contain vehicles which arepharmaceutically acceptable for formulation. These may be in particularisotonic, sterile, saline solutions (monosodium or disodium phosphate,sodium, potassium, calcium or magnesium chloride and the like ormixtures of such salts), or dry, freeze-dried compositions which uponaddition, depending on the case, of sterilized water or physiologicalsaline, permit the administration of solutions.

The compounds of the invention may be presented in the conventionalpharmacological forms of administration, such as tablets, coatedtablets, nasal sprays, solutions, emulsions, liposomes, powders,capsules or sustained release forms. Conventional pharmaceuticalexcipients as well as the usual methods of production may be employedfor the preparation of these forms.

To prepare pharmaceutical compositions, an effective amount of acompound of Formula (I) or further anticancer drug according to theinvention may be dissolved or dispersed in a pharmaceutically acceptablecarrier or aqueous medium. The compositions may comprise any knowncarrier, diluent or excipient. For example, formulations which aresuitable for parenteral administration conveniently comprise sterileaqueous solutions and/or suspensions of pharmaceutically activeingredients preferably made isotonic with the blood of the recipient,generally using sodium chloride, glycerin, glucose, mannitol, sorbitoland the like.

Excipients that may be included in any pharmaceutical compositioninclude preservatives (such as p-hydroxybenzoates), chelating agents(such as EDTA), stabilizing agents, tonicity adjusting agents,antimicrobial agents, flocculating/suspending agents, wetting agents,solvents and solvent systems, antioxidants and buffering agents, amongstothers. It is within the competencies of the person of ordinary skill inthe art to select and optimise such excipients and their amounts whenformulating a pharmaceutical composition for a particular desiredpurpose.

Compositions are preferably in the form of aqueous solutions. Suchsolutions are prepared according to known methods in the art and thenfilled into injection vials or ampoules.

The form of the pharmaceutical compositions, the route ofadministration, the dosage and the regimen naturally depend upon thenature of the cancer to be treated, the severity of the illness, theage, weight, and sex of the patient, etc., or alternatively of thedesired duration of treatment.

Treatment involves administration of a compound of Formula (I),optionally with a further anticancer agent.

The compounds of Formula (I) for use in accordance with the presentinvention may be administered to a subject via any appropriate route.The same applies to compositions or formulations comprising thecompounds of Formula (I).

The compounds of Formula (I), and therefore compositions andformulations comprising the same, may be presented, for example, in aform suitable for oral, nasal, parenteral, intravenal, topical, rectalor intrathecal administration. Preferably, the compounds are presentedin a form suitable for systemic (e.g. intravenous) administration.

Any mode of administration common or standard in the art may be used,e.g. injection, infusion, topical administration, inhalation,transdermal administration, both to internal and external body surfacesetc. by any suitable method known in the medicinal arts. Thus modes ofadministration include oral, nasal, enteral, rectal, vaginal,transmucosal, topical, or parenteral administration or by inhalation.Administration may be direct to the tumour (intratumoraladministration).

Oral or parenteral administration is preferred. Preferred parenteralmeans of administration are intravenous, intramuscular, intraperitoneal,intracranial and subcutaneous administration, and administration to thecerebrospinal fluid (intrathecal administration). More preferably, theadministration is intraperitoneal or intravenous administration, mostpreferably intravenous administration.

Preferably, the administration is oral or intravenous.

Intravenous administration may be intravenous injection or intravenousinfusion, most preferably intravenous infusion (e.g. by infusion pump).

The compound of Formula (I) and the further anticancer agent may beadministered by the same or different routes.

As noted above, the compound of Formula (I) and further anticancer agentmay be administered simultaneously, separately or sequentially. In apreferred embodiment the compound of Formula (I) and further anticanceragent are administered sequentially, e.g. at separate times, i.e. nottogether in the same composition. In an alternate embodiment thecompound of Formula (I) and further anticancer agent are administeredtogether at the same time, for example in the same composition or inseparate compositions. The timing of the separate administrations may bedetermined according to the particular compound of Formula (1) or theparticular further anticancer agent, formulations and/or modes ofadministration used. Thus the compound of Formula (I) may beadministered before or after the further anticancer agent.

For example the further anticancer agent may be administered first andthe compound of Formula (I) may be administered at a suitable timeinterval afterwards to align with the optimum time of further anticanceragent delivery to the target site, or vice versa. Such determinationsare entirely within the routine skill of the clinician. Thus for examplethe compound of Formula (I) may be administered, preferablyparenterally, more preferably intravenously at least or up to 20, 30,40, 50, 60, 70, or 90 minutes or 2, 3, 4, 5 or 6 hours before or afterthe further anticancer agent.

Doses and dosages may be determined in a routine manner and may dependupon the nature of the molecule, purpose of treatment, age of patient,mode of administration etc. Any therapeutic agent of the invention asabove described may be combined with pharmaceutically acceptableexcipients to form therapeutic compositions. A dose refers to aspecified amount of medication taken at one time, i.e. the terms “singledose” and dose are used interchangeably. A course of treatment maycomprise multiple doses, i.e. multiple single doses, over a period oftime.

In the methods and uses of the invention, preferably an effective amountof the compound of Formula (I), and the optional further anticanceragent if present, is administered. In other words, a dose preferablycomprises an effective amount of the compound of Formula (I), and theoptional further anticancer agent if present.

As shown in the Examples, the compounds of Formula (I) are tolerated athigh doses compared to the dose required to kill a tumour. This propertydiffers from many chemotherapeutic compounds; indeed, the mostchemotherapeutic compounds have substantial side effects at the doserequired to kill the cancer. The present Examples indicate that,advantageously, compounds of Formula (I) can be administered at dosesthat are far in excess of the dose necessary to kill the tumor.

As shown in the Examples, mice tolerated single doses of compounds ofFormula (I) of 300 mg/kg and 2000 mg/kg, but did not tolerate a singledose of 8000 mg/kg. These data suggest that in mice the maximumtolerated dose of the compounds of Formula (I) is at least 2000 mg/kgbut less than 8000 mg/kg. The conversion between a mouse dose and ahuman dose is a factor of 0.081 (Nair et al., (2016) Basic Clin Pharm.7(2): 27-31. The data in the Examples therefore indicates that inhumans, the maximum tolerated dose of the compounds of Formula (I) is atleast 162 mg/kg but less than 648 mg/kg.

Thus, preferably, the compound of Formula (I) is administered at a doseof ≤405 mg/kg, preferably ≤324 mg/kg, more preferably ≤243 mg/kg, morepreferably ≤162 mg/kg, more preferably ≤81 mg/kg, more preferably ≤40.5mg/kg, more preferably ≤24.3 mg/kg. Preferably, the compound of Formula(I) is administered at a dose of at least 10 mg/kg, more preferably atleast 20 mg/kg, more preferably at least 30 mg/kg, more preferably atleast 40 mg/kg, more preferably at least 500 mg/kg, more preferably atleast 100 mg/kg.

Preferably the compound of Formula (I) is administered at a dose between10 mg/kg and 405 mg/kg, preferably between 20 mg/kg and 324 mg/kg, morepreferably between 20 mg/kg and 243 mg/kg, more preferably between 30mg/kg and 162 mg/kg, more preferably between 40 mg/kg and 81 mg/kg.

These doses are preferred doses to human subjects.

Dosages, and dosage regimens, may vary based on parameters such as theage, weight, condition and sex of the subject, the purpose of treatment,the disease being treated, the age and/or condition of the patient, themode of administration etc.

Appropriate dosages and regimens can be readily established. Appropriatedosage units can readily be prepared. Dosing regimens may be determinedin a routine manner

Treatment may comprise a single administration of the compound ofFormula (I), optionally with a further anticancer agent, or may compriserepeated administrations of the compound of Formula (I), optionally witha further anticancer agent. The dosing regimen of the compound ofFormula (I) and the further anticancer agent, if present, need not beidentical. Alternatively, treatment may comprise a single administrationof the compound of Formula (I) and repeated administrations of thefurther anticancer agent, or vice versa.

Preferably, the compound of Formula (I) is administered, preferably atany one of the above described doses, every 1, 2, 3, 4, 5 or 6 days,more preferably every 2, 3, or 4 days, more preferably every 3 days fora total of 2 to 10, more preferably 3 to 8, more preferably 4 to 6, morepreferably 5 administrations.

However, it will be within the competencies of the person of ordinaryskill in the art to determine the appropriate dosing regimen, and therelevant doses therein, based upon the nature of the compound, thepurpose of treatment, the disease being treated, the age and/orcondition of the patient, the mode of administration etc.

The present invention also provides a product or kit comprising acompound of Formula (I) and a further anticancer agent. The kit orproduct can be used in any of the uses or methods described herein, i.e.for use in treating or preventing cancer. Particularly, the kit orproduct is for simultaneous, separate or sequential use. Preferably thecompound of Formula (I), and optionally also the further anticanceragent, is formulated for parenteral administration, preferably i.v.administration.

Each component of the kits of the present invention (i.e. eachanti-cancer agent) may be provided in a separate compartment or vessel.Where convenient and practical, mixtures of components could beprovided. The components may be provided in dry, e.g. crystallised,freeze dried or lyophilised, form or in solution, typically such liquidcompositions will be aqueous and buffered with a standard buffer such asTris, HEPES, etc.

Preferably the kits are for use in treating cancer, e.g. are for use inthe methods or uses of the present invention as described herein.

The compounds of the invention (i.e. the compounds of Formula (I),(IIa), (IIb), (IIIa), (IIIb), (IIIc), (IIId), (Iva), (IVb), (IVc),(IVd), (IVe) and(IVf)) are either commercially available, are known inthe literature, or may be obtained by conventional synthetic procedures,in accordance with standard techniques, from available startingmaterials using appropriate reagents and reaction conditions. In thisrespect, the skilled person may refer to inter alia “ComprehensiveOrganic Synthesis” by B. M. Trost and I. Fleming, Pergamon Press, 1991and “Protective Groups in Organic Synthesis”, 3rd edition, T. W. Greene& P. G. M. Wutz, Wiley-Interscience (1999).

The compounds of the invention are available commercially, for examplefrom Berry and Associates, Toronto Research Chemicals, Sigma Aldrich,Carbosynth, Trilink Biotech and other well-known commercial suppliers.

The invention will be further described with reference to the followingnon-limiting Examples in which:

Figure 1 shows that 5-formyl-2′-deoxycytidine and5-hydroxymethylcytidine are well tolerated in mice and reduce humanglioblastoma multiforme tumors in mouse xenograft models.

FIG. 1 A: Single dose maximum tolerated dose protocol. Mice were dosedwith a single intraperitoneal injection at the indicated dose. If allthe mice in the relevant group tolerated the indicated dose, the dosewas escalated as indicated.

FIG. 1 B: Mice body weigh was measured at the indicated time point aftermice were intraperitoneally injected with the indicated dose andindicated compound every three days for a total of five doses.

FIGS. 1C-H: U87-MG cells were implanted in the flank of 32immunodeficient mice. After tumors reached 129-131 mm³, mice weredivided into four groups of 8 mice. The negative control group wastreated with the vehicle, the positive control group was treated with 40mg/kg temozolomide once a day for five days, the treatment groups weretreated with 2000 mg/kg 5-formyl-2′-deoxycytidine or 2000 mg/kg5-hydroxymethyl-2′-deoxycytidine once every three days for a total offive doses. Tumor volumes were measured every three days (FIG. 1C) andthe mouse body weight was measured every three days (FIG. 1 D). At thecompletion of the study the percent tumor growth inhibition was computed(TGI(%)) (FIG. 1 E), tumors were resected, photographed (FIG. 1F),measured (FIG. 1G) and sectioned and stained with hematoxylin and eosin(FIG. 1H).

FIG. 2 shows that 5-formyl-2′-deoxycytidine and5-hydroxymethyl-2′-deoxycytidine kill glioblastoma multiforme by amechanism unrelated to current nucleotide analogues:

FIG. 2A: flow cytometry of 5-formyl-2′-deoxycytidine and5′-hydroxymethyl-2′-deoxycytidine treated cells, stained with Annexin Vand 7AAD.

FIG. 2B: Survival curves of HeLa cells treated with a titration of5-formylcytosine, 5-formylcytidine, or 5-formyl-2′-deoxycytidine.

FIG. 2C: Survival curves of U87-MG cells treated with a titration5-hydroxymethylcytosine, 5-hydroxymethylcytidine, or5-hydroxymethyl-2′-deoxycytidine.

FIG. 3 shows quantification of mutations in the Hypoxanthine-guaninephosphoribosyltransferase (HPRT) gene induced by5-formyl-2′-deoxycytidine and 5-hydroxymethyl-2′-deoxycytidine inmammalian cells. 5-formyl-2′-deoxycytidine and5-hydroxymethyl-2′-deoxycytidine are shown not to be mutagenic.

Figure 4 shows the level of cytotoxicity (% survival) of5-formyl-2′-deoxycytidine, 5-formylcytidine and5-chloro-2′-deoxycytidine against HeLa cells after three days oftreatment.

FIG. 5 shows the level of cytotoxicity (% survival) of5-formyl-2′-deoxycytidine (d5fC), 5-hydroxymethyl-2′-deoxycytidine(d5hmC), 5-chloro-2′-deoxycytidine (5CldC), 5-bromo-2′-deoxycytidine(5BrdC), 5-lodo-2′deoxycytidine (51dC), and Thymidine vs. U87-MG cells.

FIG. 6 shows that the cytotoxic effects(% survival) of5-formyl-2′-deoxycytidine and 5-hydroxymethyl-2′-deoxycytidine are notrescued by the addition of thymidine in U87-MG cells, indicating that5-formyl-2′-deoxycytidine and 5-hydroxymethyl-2′-deoxycytidine do notact by inhibiting thymidine synthase.

FIG. 7 shows the cytotoxic effects (% survival) of5-formyl-2′-deoxycytidine in combination with Temozolomide in U87-MGcells.

FIG. 8 shows the cytotoxic effects (% survival) of5-hydroxymethyl-2′-deoxycytidine in combination with Temozolomide inU87-MG cells.

FIG. 9 shows the cytotoxic effects of 5-methoxymethyl-2′-deoxyuridineand 5-acetoxymethyl-2′-deoxyuridine on U87-MG cells. Treatment for 72hrs. Survival was quantified using an MTT assay.

FIG. 10 shows % survival of various cells after treatment with d5fCTP ord5hmCTP

FIG. 10A: % survival of HeLa after treatment with5-formyl-2′-deoxycytidine-5′-triphosphate (d5fCTP) for 72 hrs. Survivalwas quantified using an MTT assay.

FIG. 10B: % survival of U87-MG cells (Glioma, Grade IV) after treatmentwith either 5-formyl-2′-deoxycytidine-5′-triphosphate or5-hydroxymethyl-2′-deoxycytidine-5′-triphosphate for 72 hours. Survivalwas quantified using an MTT assay.

FIG. 11 shows the CDA expression levels in various cell lines. Thevalues are normalized Log 2 CDA expression levels. The expression levelswere determined using the Affymetrix Human Genome U133 Plus 2.0 Arrayplatform and the Genevestigator database(https://genevestigator.com/gv/).

FIG. 12 shows the linear CDA expression levels in various cell lines.The expression levels were determined using the Affymetrix Human GenomeU133 Plus 2.0 Array Platform and the Genevestigator database(https://genevestigator.com/gv/).

FIG. 13A and 13B show the CDA expression levels in various human braintumours. The values are normalized Log 2 CDA expression levels. Theexpression levels were determined using the Affymetrix Human Genome U133Plus 2.0 Array platform and the Genevestigator database(https://genevestigator.com/gv/).

FIGS. 14A and 14B show the linear CDA expression levels in various humanbrain tumours. The expression levels were determined using theAffymetrix Human Genome U133 Plus 2.0 Array platform and theGENEVESTIGATOR® database (https://genevestigator.com/gv/).

FIG. 15 shows the results of a PAMPA Assay demonstrating that 2d5hmC and2d5fC can pass the blood-brain barrier.

EXAMPLES Materials and Methods Animals

All aspects of this work, including housing, experimentation, anddisposal of animals were performed in general accordance with the Guidefor the Care and Use of Laboratory Animals: Eighth Edition (NationalAcademy Press, Washington, D.C., 2011) in an AAALAC-accreditedlaboratory animal facility. The animal care and use protocol wasreviewed and approved by the IACUC at Pharmacology Discovery ServicesTaiwan, Ltd.

Cell Culture

Primary glioma neural stem (GNS) cells (G7, G14, G144, G166) werecultured on poly-D-lysine (Merck Millipore, Cat. Nr. A-003-E) andlaminin (R&D Systems, Cat.Nr. 3446-005-01) coated plates in neural stemcell medium (50% DMEM-F12 (Thermofisher, Cat. Nr. 21041025), 50%neurobasal medium (Thermofisher, Cat. Nr. 10888-022), N2 (LifeTechnologies, Cat. Nr. A-003-E) and B27 supplements (Life Technologies,Cat.Nr. 12587010), 1 mM sodium pyruvate (Life Technologies, Cat.Nr.11360-039), 2 mM glutamax (Life Technologies, Cat. Nr. 35050038), 1 mMHEPES (Fisher Scientific, Cat. Nr. BP299-1), 0.1 mM p-mercaptoethanol(Life Technologies, Cat. Nr. 31350010), 1× nonessential amino acids(Life Technologies, Cat. Nr. 11140-035), 0,006% bovine serum albumin(Sigma, Cat. Nr. A8577-10ML),4 μg/ml heparin (Sigma, Cat. Nr.H3149-25KU), 100 U/ml penicillin, 100 μg/ml streptomycin, 20 ng/ml hEGF(R&D Systems, Cat. Nr. 236-EG-200), 10 ng/ml bFGF (Peprotech, Cat. Nr.100-18B).

HCoT16 were grown in McCoAs 5a Medium modified (Life technologies,Cat.Nr. 36600021) supplemented with 10% Fetal Bovine Serum and 100 U/mipenicillin 100 U/ml streptomycin.

Arpe 19 were grown in DMEM:F12 medium (Life Technologies, 21331-020)supplemented with 10% Fetal Bovine Serum and 100 U/m penicillin 100 U/m5streptomycin.

HAP1 were grown in IMDM (Gibco, Cat. Nr. 12440-05) supplemented withsupplemented with 10% Fetal Bovine Serum and 100 U/m penicillin 100 U/m5streptomycin.

Cell lines not mentioned above were grown in DMEM (Sigma, Cat.Nr. 06429)supplemented with 10% Fetal Bovine Serum and 100 U/mA penicillin 100U/m5 streptomycin. All cells were maintained in a 5% 002, humidified,water-jacketed incubator at 37° C. Cells were passed between 70 and 90%confluence.

Drugs

Compounds used in the present Examples were obtained as follows (CAT#0catalogue number).

Compound CAS # Source CAT # 5-hydroxymethyl-2′-deoxycytidine 7226-77-9Berry and Associates PY 7588 5-formyl-2′-deoxycytidine 137017-45-9 Berryand Associates PY 7589 5-carboxyl-2′-deoxycytidine 1009808-62-1 Berryand Associates PY 7593 5-formylcytidine 148608-53-1 Berry and AssociatesPY 7599 5-formylcytosine 4425-59-6 Toronto Research F698975 ChemicalsTemozolomide 85622-93-1 Sigma Aldrich T2577 5-flurouracil 51-21-8 SigmaAldrich F6627 5-hydroxymethylcytidine 19235-17-7 Berry and Associates PY7596 5-hydroxymethylcytosine 1123-95-1 Toronto Research H945870Chemicals 5-chloro-2′-deoxycytidine 32387-56-7 Carbosynth NC082795-bromo-2′-deoxycytidine 1022-79-3 Carbosynth NB064505-iodo-2′-deoxycytidine 611-53-0 Carbosynth ND05777 Thymidine 50-89-5Sigma Aldrich T1895 5-methoxymethyl-2′- 5116-22-3 Toronto ResearchM263610 deoxyuridine Chemicals 5-acetoxymethyl-2′-deoxyuridine148380-55-6 Toronto Research A167180 Chemicals5-formyl-2′-deoxycytidine-5′- Trilink Biotech N-2064 triphosphate5-Hydroxymethyl-2′-deoxycytidine-5′-Triphosphate Trilink Biotech N-2060

Survival Assay

In a 96-well plate, 4000 cells in 100 μl of corresponding medium wereseeded. The following day, drugs, diluted in DMSO, were added in 8concentrations in triplicate. Drugs were added as a 4-fold dilutionseries starting from 100 μM. Cells were incubated with the drugs for 72hours. Cell proliferation was assessed by MTT assay according to themanufacturer's protocol (ATCC, Cat. Nr. 30-1010K). Cell survival wasnormalized to the survival of cells treated with DMSO only. Experimentwas performed three times, data represent mean of nine wells±SEM.

MTD (Maximum Tolerated Dose)

5-hydroxymethyl-2′-deoxycytidine and 5-formyl-2′-deoxycytidine (Berryand Associates), were formulated in dimethyl sulfoxide (DMSO)/Solutol® RHS15/phosphate buffered saline (PBS) (5/5/90, v/v/v) at theconcentration of 30 and 200 and 400 mg/mL for IP administration at thedosing volume of 5 mL/kg. A dosing volume at 10 or 20 mL/kg was applied.

Male ICR mice weighing 23±3 g were provided by BioLasco Taiwan (underCharles River Laboratories Licensee). Animals were acclimated for 3 daysprior to use and were confirmed with good health. All animals weremaintained in a hygienic environment with controlled temperature (20-24°C.), humidity (30%-70%) and 12 hours light/dark cycles. Free access tosterilized standard lab diet [MFG (Oriental Yeast Co., Ltd., Japan)] andautoclaved tap water were granted.

5-hydroxymethyl-2′-deoxycytidine and 5-formyl-2′-deoxycytidine wereadministered IP to groups of three male ICR mice weighing 23±3 g.Animals received an initial dose of 300 mg/kg. If the animals survivedfor 72 hours, the dose for the next cohort was increased. If one or moreanimals died, the dose for the next cohort was decreased. The testingstopped when all animals survived at the upper bound, or when three doselevels had been tested or when the upper or lower bound had beenreached. At each dose level, animals were observed for the presence ofacute toxic symptoms (mortality, convulsions, tremors, musclerelaxation, sedation, etc.) and autonomic effects (diarrhea, salivation,lacrimation, vasodilation, piloerection, etc.) during the first 30minutes, again at 1, 24, 48 and 72 hours. Body weights were recordedpre-dose and at 72 hours. The animals were observed and mortality noteddaily after compound administration. Gross necropsy was performed on allanimals without tissue collection, and the next dose level wasdetermined based on the study design table.

Multiple MTD

5-hydroxymethyl-2′-deoxycytidine and 5-formyl-2′-deoxycytidine (Berryand Associates) were formulated in dimethyl sulfoxide (DMSO)/Solutol® RHS15/phosphate buffered saline (PBS) (5/5/90, v/v/v) at theconcentration of 15, 50, and 100 mg/mL for IP administration at thedosing volume of 20 mL/kg. The test compounds were dosed every threedays for a total of 5 doses (q3d×5).

Male ICR mice weighing 23±3 g were provided by BioLasco Taiwan (underCharles River Laboratories Licensee). Animals were acclimated for 3 daysprior to use and were confirmed with good health. All animals weremaintained in a hygienic environment with controlled temperature (20-24°C.), humidity (30%-70%) and 12 hours light/dark cycles. Free access tosterilized standard lab diet [MFG (Oriental Yeast Co., Ltd., Japan)] andautoclaved tap water were granted.

Animals were observed for the presence of acute toxic symptoms(mortality, convulsions, tremors, muscle relaxation, sedation, etc.) andautonomic effects (diarrhea, salivation, lacrimation, vasodilation,piloerection, etc.) during the first 30 minutes after each treatment(Days 1, 4, 7, 10 and 13), and again at 1, 24, 48 and 72 hours after thefinal dose (Day 13). The mortality was noted at the same scheme. Inaddition, body weights were recorded before each treatment and at 24, 48and 72 hours after the final administration. Gross necropsy wasperformed on all animals without tissue collection.

Xenograft

5-hydroxymethyl-2′-deoxycytidine and 5-formyl-2′-deoxycytidine (Berryand Associates) dosing solutions were prepared fresh prior to each doseadministration by first adding appropriate volume of DMSO to pre-weighedcompound, then adding appropriate volumes of Solutol® and PBS (5%DMSO/5% Solutol®/90% PBS). Standard agent, Temozolomide, was provided byOslo University Hospital in powder form and was formulated fresh priorto each dose by first adding appropriate volume of DMSO to pre-weighedcompound, then adding appropriate volumes of Solutol® and PBS (5%DMSO/5% Solutol®/90% PBS). 5-hydroxymethyl-2′-deoxycytidine and5-formyl-2′-deoxycytidine were administered at a dose volume of 20mL/kg. Standard agent, Temozolomide, was administered at a dose volumeof 10 mL/kg.

The human brain malignant glioma cell line, U87-MG (ATCC HTB-14,epithelial glioblastoma), was obtained from American Type CultureCollection (ATCC). The cells were cultured in Minimum essential mediumcontaining 5% fetal bovine serum (FBS) at 37° C., with 5% CO2 in anincubator.

Female (nu/nu) nude mice aged 6-7 weeks obtained from BioLasco Taiwan(under Charles River Laboratories Licensee) were used. The animals werehoused in individually ventilated cages (IVC, 36 Mini Isolator system).The allocation for 5 animals was 27×20×14 in cm. All animals weremaintained in a hygienic environment under controlled temperature(20-24° C.) and humidity (30%-70%) with 12-hour light/dark cycle. Freeaccess to standard lab diet [MFG (Oriental Yeast Co., Ltd., Japan)] andautoclaved tap water were granted.

Viable U87-MG (ATCC HTB-14) cells were subcutaneously (SC) implanted(5×106 cells/mouse in PBS at 0.2 mL/mouse) into the right flank offemale nu/nu mice.

When group mean tumor volumes reached approximately 129 mm³ to 131 mm³,tumor implanted mice were divided into four treatment groups, each groupcontaining eight animals, and dose administrations were initiated(denoted as Day 1).

5-hydroxymethyl-2′-deoxycytidine, 5-formyl-2′-deoxycytidine at 2000mg/kg and corresponding vehicle (5% DMSO/5% Solutol®/90% PBS) wereadministered intraperitoneally (IP) once every three days for a total offive administrations. Temozolomide at 40 mg/kg was administered orally(PO) once daily for five total administrations.

The tumor volume, body weight, mortality, and signs of overt toxicitywere monitored and recorded twice weekly for 29 days. Tumor volume (mm³)was estimated according to the ellipsoid formula as:length×(width)²×0.5. Tumor growth inhibition (T/C) was calculated by thefollowing formula:

%  T/C = (Tn/Cn) × 100%

-   -   Cn: Tumor weight measured on Day n in the control group    -   Tn: Tumor weight measured on Day n in the treated group    -   % T/C value≤42% was considered significant antitumor activity        (#).        Percent tumor growth inhibition (TGI) was also calculated by the        following formula:

%  THI = (1 − (Tn/Cn)) × 100%

-   -   % TGI value≥58% was considered significant antitumor activity        (#).

Two-way ANOVA® followed by Bonferroni test was also used to ascertainthe statistically significant difference compared to the negativecontrol group during study; Day 1 through to Day 29. Differences areconsidered significant at p<0.05 (*).

Upon study completion, tumors were excised from all animals on study andphotographs were taken.

HPRT Assay

HPRT mutagenicity assay was performed in V79 cells. 50,000 V79 cellswere treated with three different concentrations of either d5hmC or d5fC(1, 10, 100 μM) for 24 hours in a 6 well plate. DMSO was used as anegative control. After the treatment, the cells were subcultured asneeded in T75 flasks for 9 days to allow expression of HPRT-mutants.10,000 cells were replated into 10 replica Petri dishes (100×15 mm) withselective media (2.5 μg/ml of 6TG).

Survival (relative plating efficiency) was determined by plating 200cells into four replica Petri dishes (60×15 mm) without selective media.Colonies were fixed, Giemsa-stained and counted 7 days later. The mutantfrequency is expressed as a total number of mutants counted on allplates divided by the number of cells seeded corrected by reseedingplating efficiency. Experiment was performed three times, data representmean of triplicate±SEM.

Apoptosis Flow Cytometry

100,000 cells were seeded in a 6-well plate and incubated with 100 μM ofTemozolomide, 2′-Deoxy-5-hydroxymethylcytidine,5-Formyl-2′-deoxycytidine or DMSO for 72 hours. Detection of apoptoticcells was assessed using Annexin V-7-amino-actinomycin D (7-AAD)Apoptotic Detection Kit (Nordic Biosite AS, Cat. Nr. 640922) accordingto manufacturer's protocol.

Fluorescence-activated cell sorting analysis was performed on LSRFortessa (BD Biosciences) and data were analyzed on FlowJo software. Allexperiments were performed in triplicates.

Western Blots

Western blots were performed as previously described (Towbin et al.,1979. Biotechnology, 24, 145-149). Anti-CDA antiserum (Abcam, cat nr.Ab82346) was used according to the manufacturer's recommendedconcentration. Proteins were quantified by the signal generated by theoxidation of luminol by horse radish peroxidase conjugated to thesecondary antibody.

Example 1: Cytotoxicity to Tumour Cells

HeLa cells, grown as described above (Materials and Methods), weretreated with 5-hydroxymethyl-2′-deoxycytidine and5-formyl-2′-deoxycytidine. After three days of treatment with thesecompounds, cell survival was assessed as described above (Materials andMethods). While survival after treatment with5-hydroxymethyl-2′-deoxycytidine did not differ from DMSO treated cells,it was found, surprisingly, that 5-formyl-2′-deoxycytidine was cytotoxicto HeLa cells.

The cytotoxic effect of that 5-formyl-2′-deoxycytidine was compared totwo well-described cytotoxic compounds, 5-flurouracil and temozolamide.5-formyl-2′-deoxycytidine was determined to be more cytotoxic to HeLa(IC50=0.76 μM) cells than both 5-flurouracil (IC50>25 μM) andtemozolomide (IC50>25 μM).

With the knowledge that 5-formyl-2′-deoxycytidine is cytotoxic tocervical carcinoma (HeLa) cells, the study was expanded in twodirections: (i) a further compound was assessed:5-carboxyl-2′-deoxycytidine; and (ii) their cytotoxic effects against awide range of human cancer cell lines was assessed (Table 1).

TABLE 1 Evaluation of 5-formyl-2′-deoxycytidine (2d5fC),5-hydroxymethyl-2′-deoxycytidine (2d5hmc) and 5-carboxy-2′-deoxycytidine (2d5caC) in a range of human cancer cell lines, and ascompared to Temozolomide and 5-flurouracil (5fU). IC₅₀ is theconcentration at which half of the cells are killed by the relevantcompound. Cell Germ IC₅₀ (μM) Line Disease Layer 2d5fC 2d5hmC 2d5caCTemozolomide 5fU U87-MG Glioma, Grade IV Ectoderm 0.33403.027 >25.00 >25.00 >25.00 HCT-116 Colon CarcinomaEndoderm >25.00 >25.00 >25.00 >25.00 3.545 A549 Lung CarcinomaEndoderm >25.00 >25.00 >25.00 1.018 1.853 22Rv1 Prostate CarcinomaEndoderm >25.00 >25.00 >25.00 >25.00 >25.00 NCI-N87 Gastric CarcinomaEndoderm 0.8057 >25.00 >25.00 >25.00 1.524 MIA PaCa-2 PancreaticEndoderm 1.143 >25.00 >25.00 >25.00 8.096 A-498 Kidney CarcinomaMesoderm >25.00 >25.00 >25.00 >25.00 2.367 U937 Lymophoma Mesoderm0.9536 >25.00 >25.00 13.08 7.449 A375 Malignant Melanoma Mesoderm3.188 >25.00 >25.00 >25.00 7.867 HL-60 Acute Promyelocytic Mesoderm6.262 >25.00 >25.00 >25.00 >25.00 Leukemia SK-OV-3 Ovarian Mesoderm9.690 >25.00 >25.00 >25.00 3.756 Adenocarcinoma MCF-7 EpithelialMesoderm 0.8657 >25.00 >25.00 >25.00 >25.00 Adenocarcinoma (Breast) U2OSBone Mesoderm 8.69 >25.00 >25.00 >25.00 2.889 HeLa Cervical Mesoderm0.76 >25.00 >25.00 >25.00 >25.00 Adenocarcinoma HAP1 ChronicMesoderm >25.00 6.53 n.d. n.d. n.d. Myelogenous Leukemia (CML) V79Chinese Hamster Mesoderm >25.00 >25.00 n.d. n.d. n.d. Ovary H1437 Lung(metastatic Endoderm 5.65 n.d. n.d. n.d. n.d. non-small celladenocarcinoma) H1573 Lung (metastatic Endoderm 7.72 n.d. n.d. n.d. n.d.adenocarcinoma)

As shown in Table 1, 5-carboxyl-2′-deoxycytidine had no cytotoxicproperties in any of the cancer cell lines evaluated. Interestingly,5-formyl-2′-deoxycytidine is cytotoxic to a wide range of human cancercells, indicating its potential use in the treatment of a wide range ofcancers. 5-hydroxymethyl-2′-deoxycytidine has a narrower cytotoxicityprofile; indeed this cytidine derivative was only cytotoxic to two celllines evaluated: U87-MG, grade IV glioma cells (IC50>0.3340 μM) and HAP1Chronic Myelogenous Lukemia cells (IC50>3.027 μM). This suggests thatthis compound may be well tolerated by patients.5-formyl-2′-deoxycytidine and 5-hydroxymethyl-2′-deoxycytidine wereobserved as the most cytotoxic to glioblastoma multiforme cell (U87-MG)lines.

Since the greatest cytotoxic effect of 5-formyl-2′-deoxycytidine and5-hydroxymethyl-2′-deoxycytidine was observed in glioma grade IV(U87-MG) cells, the cytotoxic activity of these compounds was evaluatedin a wider range of patient derived grade IV, glioma cells (Table 2).Due to lack of activity in previous assays, 5-carboxyl-2′-deoxycytidinewas not included in this more rigorous analysis.

TABLE 2 Evaluation of cytotoxicity of 5-formyl-2′-deoxycytidine (2d5fC)and 5-hydroxymethyl- 2′-deoxycytidine (2d5hmc) against patient derivedglioblastoma multiforme cell lines (Glioma, Grade IV), and as comparedto Temozolomide and 5-flurouracil (5fU). IC₅₀ is the concentration atwhich half of the cells are killed by the relevant compound. Cell GermIC₅₀ (μM) Line Disease Layer 2d5fC 2d5hmC Temozolomide 5fU U87-MGGlioma, Grade IV Ectoderm 0.3340 3.027 >25.00 >25.00 G7 Glioma, Grade IVEctoderm 19.25 11.93 6.176 6.456 G14 Glioma, Grade IV Ectoderm >100 25.46.018 11.61 G26 Glioma, Grade IV Ectoderm 28.12 >100 7.338 7.125 G30Glioma, Grade IV Ectoderm 18 28.13 21.6 23.33 G144 Glioma, Grade IVEctoderm >100 >100 14.66 6.805 G166 Glioma, Grade IVEctoderm >100 >100 >100 4.813 SF188 Glioma, Grade IV Ectoderm 28.248.975 >100 3.884 U3017 Glioma, Grade IV Ectoderm >100 14.12 >100 3.921DIPG007 Glioma, Grade IV Ectoderm >100 >100 >100 4.302 U3013 Glioma,Grade IV Ectoderm >100 >100 >100 4.138 CB152 Glioma, Grade IVEctoderm >100 >100 >100 6.496

As shown in Table 2, after treatment with the relevant compound, 5 of 12Grade IV, Glioma cell lines were killed by 5-formyl-2′-deoxycytidine and6 of 12 Grade IV, Glioma cell lines were killed by5-hydroxymethyl-2′-deoxycytidine. This indicates the ability of thesecompounds against a broad range of Gliomas. These results werebenchmarked to both temozolomide, the current frontline treatment forGrade IV, Gliomas, and 5-flurouracil, a broad use anti-cancer drug.Temozolomide effectively killed 5 of 12 Glioma, Grade IV cell lines and5-flurouracil killed 11 of 12 Glioma, Grade IV cell lines. Thus, thedata demonstrates that 5-formyl-2′-deoxycytidine is as effective as thecurrent frontline treatment for Grade IV, Gliomas and that5-hydroxymethyl-2′-deoxycytidine is more effective than the currentfrontline treatment for Grade IV, Gliomas.

Furthermore, Table 2 demonstrates that both 5-formyl-2′-deoxycytidineand 5-hydroxymethyl-2′-deoxycytidine are cytotoxic to cell lines thatare resistant to the current frontline treatment (Temozolomide). Thisprovides evidence that these compounds may perform better than currenttreatments for such Temozolomide resistant tumours. Alternatively, Table2 indicates that temozolomide in combination with5-formyl-2′-deoxycytidine and/or 5-hydroxymethyl-2′-deoxycytidine wouldbe an optimal treatment.

Example 2: Cytotoxicity to Normal Cells

As demonstrated in Example 1, 5-formyl-2′-deoxycytidine and5-hydroxymethyl-2′-deoxycytidine are useful therapeutics for thetreatment of cancers, particularly Grade IV gliomas, and particularlythose that are resistant to temozolomide treatment.

The present inventors further investigated the extent to which thesecompounds kill normal human cells; low cytotoxicity against normal humancells is advantageous property for anti-cancer agents. The cytotoxicityof these compounds in various normal human cell lines was thereforeinvestigated (Table 3). Cells were grown and the survival assay wasperformed as described above (Materials and Methods).

TABLE 3 Evaluation of cytotoxicity of 5-formyl-2′-deoxycytidine (2d5fC)and 5-hydroxymethyl- 2′-deoxycytidine (2d5hmC) to normal (non-cancerous)human cell lines,), and as compared to Temozolomide and 5-flurouracil(5fU). IC₅₀ is the concentration at which half of the cells are killedby the relevant compound. Cell Germ IC₅₀ (μM) Line Disease Layer 2d5fC2d5hmC Temozolomide 5fU HEK293FT Normal Mesoderm,Kidney >25.00 >25.00 >25.00 Arpe19 Normal Ectoderm,ReMna >25.00 >25.00 >25.00 >25.00 HaCat Normal Mesoderm,0.1024 >25.00 >25.00 0.5228 KeraMnocyte MRC5 Normal Endoderm,Lung >25.00 >25.00 >25.00 >25.00 Buffy A Normal Mesoderm,Bone >25.00 >25.00 n.d. >25.00 Marrow Buffy C Normal Mesoderm,Bone >25.00 >25.00 n.d. >25.00 Marrow Buffy M Normal Mesoderm,Bone >25.00 >25.00 n.d. >25.00 Marrow

As shown in Table 3, 5-hydroxymethyl-2′-dexycytidine was not cytotoxicto any of the normal cell lines evaluated. 5-formyl-2′-deoxycytidine wascytotoxic only to one normal human cell line (HaCat, Keratinocytes).These results indicate that these compounds are not only effectivecancer chemotherapeutics but also can be well tolerated by humans.

Example 3: Maximum Tolerated Dose

Since 5-hydroxymethyl-2′-dexycytidine and 5-formyl-2′-deoxycytidine havelimited effects on normal cells, the present inventors considered thatthe compounds could be given at relatively high doses without causingside effects normally associated with cancer chemotherapy. The maximumtolerated dose (MTD) of these compounds in mice was determined asdescribed above (Materials and Methods). An MTD scheme was developed(FIG. 1A). While the end-point of the study was survival after 72 hours,the presence of acute toxic symptoms (mortality, convulsions, tremors,muscle relaxation, sedation, etc.) and autonomic effects (diarrhea,salivation, lacrimation, vasodilation, piloerection, etc.) in theanimals was monitored during the first 30 minutes, and again at 1, 24,48 and 72 hours. Body weights were recorded pre-dose and at 72 hours.

The results indicated that mice can tolerate a single dose of 300 mg/kgand 2000 mg/kg of 5-formyl-2′-dexycytidine and5-hydroxymethyl-2′-deoxycytidine. Mice were unable to tolerate a singledose of 8000 mg/kg 5-formyl-2′-deoxycytidine or5-hydroxymethyl-2′-deoxycytidine (not shown). These results suggest thatin mice the maximum tolerated single dose of both5-formyl-2′-deoxycytidine and 5-hydroxymethyl-2′-deoxycytidine is atleast 2000 mg/kg but less than 8000 mg/kg.

The conversion between a mouse dose and a human dose is a factor of0.081 (Nair et al., (2016) Basic Clin Pharm. 7(2): 27-31. The datatherefore indicates that in humans, the maximum tolerated single dose ofthe compounds of Formula (I) is at least 162 mg/kg but less than 648mg/kg.

Cancer chemotherapeutic drugs that can be tolerated after multiplerepetitive doses over many days are advantageous. Therefore, the presentinventors conducted a repeated maximum tolerated dose evaluation forboth 5-formyl-2′-deoxycytidine and 5-hydroxymethyl-2′-deoxycytidine asdescribed above (Materials and Methods). As they were well tolerated asa single dose in both treatment groups, 300 mg/kg, 1000 mg/kg, and 2000mg/kg doses were selected for repeated multiple tolerated doseevaluation.

Mice were injected with the indicated compound at the indicated doseintraperitoneally once every three days for a total of 5 doses. Whilesurvival was an end-point of this study, the primary end-point of thestudy was body weight; the presence of acute toxic symptoms (mortality,convulsions, tremors, muscle relaxation, sedation, etc.) and autonomiceffects (diarrhea, salivation, lacrimation, vasodilation, piloerection,etc.) in the animals was also monitored during the first 30 minutes,again at 1, 24, 48 and 72 hours. Body weights were recorded pre-dose andat 72 hours.

Body weight in untreated animals was not statistically different fromanimals treated with either 5-formyl-2′-deoxycytidine or5-hydroxymethyl-2′-deoxycytidine at any dose evaluated (FIG. 1B). Theseresults indicate that these compounds are well tolerated at theindicated doses over extended periods of time.

Example 4: In Vivo Cytotoxicity

5-formyl-2′deoxycytidine and 5-hydroxymethyl-2′-deoxycytidine wereevaluated in a Glioblastoma Multiforme mouse xenograft model asdescribed above (Materials and Methods). U87-MG cells were injectedsubcutaneously into the flank of nude mice. Tumours were allowed to formas described above (Materials and Methods). After the tumours reachedbetween 129 mm³ and 131 mm³ animals were divided into 4 groups—twotreatment groups, a negative control group and a positive control group.The two treatment groups were 5-formyl-2′-deoxycytidine and5-hydroxymethyl-2′-deoxycytidine. Mice in the treatment groups receivedone 2000 mg/kg dose of either 5-formyl-2′-deoxycytidine or5-hydroxymethyl-2′-deoxycytidine every three days for a total of 5doses. The negative control group was treated identically to thetreatment group except the IP injection contained vehicle and nocompound. The positive control group was treated with 5 daily doses of40 mg/kg temozolomide.

The tumour volume (FIG. 1C), body weight (FIG. 1D), mortality, and signsof overt toxicity were monitored and recorded twice weekly for 29 days.Tumour volume (mm³) was estimated according to the ellipsoid formula as:length×(width)²×0.5. Percent tumour growth inhibition (% TGI) wasdetermined using the following formula: % TGI=(1−[(Tn)/(Cn)])×100, whereTn=mean tumour volume of treated group on day “n”, and Cn=mean tumourvolume of control group on day “n”. A % T/C value≤42% or a percent TGIvalue≥58% compared to that of the negative control group was consideredsignificant anti-tumour activity. Two-way ANOVA® followed by Bonferronitest was also used to ascertain the statistically significant differencecompared to the negative control group during the study; Day 1 throughto Day 29 (*p<0.05).

FIG. 1C indicates that treatment with 5-formyl-2′-deoxycytidine or5-hydroxymethyl-2′-deoxycytidine resulted in marked decrease in tumourvolume, comparable to that achieved with Temozolomide, over all timepoints.

At the end of the study, both 5-formyl-2′-deoxycytidine and5-hydroxymethyl-2′-deoxycytidine showed significant anti-tumouractivity, 83% and 93% TGI respectively (FIG. 1E). The positive controlgroup treated with temozolomide showed 94% TGI. All the compounds werewell tolerated by the mice and no significant changes in body weightwere observed as a result of the treatment (FIG. 1D). At the completionof this study tumours were dissected from the mouse and photographed(FIG. 1F) and measured (FIG. 1G); marked cytotoxic effects were observedwith both of the treated groups.

One mouse in the control group died during this experiment the tumourvolume of this mouse was not reported on day 29; however, tumour volumesof this mouse are included for the prior days.

Together, these surprising results demonstrate that5-formyl-2′-deoxycytidine and 5-hydroxymethyl-2′-deoxycytidineefficiently kill Glioblastoma Multiforme cells (Glioma, Grade IV) andare not strongly cytotoxic to normal cells. Furthermore, these compoundsare well tolerated in mice, and achieve marked reduction in tumourvolume with minimal side effects in mice. The results demonstrate thatthe compounds of the invention can be used to treat human cancers, andthey can be used at high doses to kill tumour cells without killingnon-cancerous cells. Thus, there is a wide therapeutic window for theuse of these compounds as anti-cancer drugs.

Example 5: Cytotoxic Vs. Cytostatic Effect

The above cell line assays measure metabolic activity, and as such donot distinguish between inhibition of cell division and cell death. Itwas therefore evaluated whether 5-formyl-2′deoxycytidine and5-hydroxymethyl-2′-deoxycytidine were killing sensitive cells or causingthem to be delayed in the cell cycle. SF-188 grade IV glioma cells weretreated either with DMSO, temozolomide, 5-formyl-2′-deoxycytidine, or5-hydroxymethyl-2′-deoxycytidine at the indicated concentration. Cellswere harvested and stained with Annexin V (detects apoptotic cells byits ability to bind to phosphatidylserine) and 7AAD (a DNA stain whichdoes not readily pass through intact cell membranes; cells withcompromised membranes will therefore be selectively stained) andanalyzed by flow cytometery as described above (Materials and Methods).SF-188 cells treated with DMSO or temozolomide yielded similarcytometric profiles with a slight increase in the amount of dead cellsin the temozolomide treated control. A majority of SF-188 cells treatedwith 5-formyl-2′-deoxycytidine or 5-hydroxymethyl-2′-deoxycytidine weredead according to the flow cytometry profile (FIG. 2A). It is clear thatSF188 cells exposed to 5-formyl-2′-deoxycytidine or5-hydroxymethyl-2′-deoxycytidine are dead and not arrested at a cellcycle checkpoint.

Example 6: Requirement for 2′-Deoxy Sugar Derivatives

5-flurouracil, a widely used cancer chemotherapeutic drug, has multiplevariants that are cytotoxic; these variants include the nucleoside(5-flurouridine and 5-fluro-2′-deoxyuridine) and nucleobase5-flurouracil. The present inventors evaluated whether theribonucleoside and nucleobase variants of 5-formyl-2′-deoxycytidine and5-hydroxymethyl-2′-deoxycytidine would also be cytotoxic.

As shown in Table 1, 5-formyl-2′-deoxycytidine is cytotoxic to HeLacells. The cytotoxicity of 5-formylcytidine and 5-formylcytosine in HeLacells was evaluated in a survival assay as described above.Surprisingly, and in contrast to 5-fluorouridine and 5-fluorouracil,neither 5-formylcytidine nor 5-formylcytosine was cytotoxic to HeLacells (FIG. 2B).

Table I shows that 5-hydroxymethyl-2′-deoxycytidine is cytotoxic toU87-MG cells. Cytotoxicity of the 5-hydroxymethylcytidine and5-hydroxymethylcytosine in U87-MG cells was evaluated in a survivalassay as described above. Surprisingly, and in contrast to5-fluorouridine and 5-fluorouracil, 5-hydroxymethylcytidine and5-hydroxymethylcytosine were shown not to be cytotoxic to U87-MG cells(FIG. 2C).

These results indicate that the 2′-deoxyribose sugar is necessary forcytotoxicity of the compounds of the present invention. In turn, thisresult indicates, surprisingly, that 5-formyl-2′-deoxycytidine and5-hydroxymethyl-2′-deoxycytidine exploit a fundamentally differentcellular pathway than fluropyrimidines.

Example 7: Non-Effect of Cytidine Deaminase

Mutated nucleoside and nucleotide analogues are often removed from thenucleotide pool by cytidine deaminase (CDA). CDA inactivates gemcitabineand cytosine arabinoside—two common nucleotide analogue anti-canceragents. Anti-cancer agents that are not inactivated by CDA would bedesirable. Since 5-formyl-2′-deoxycytidine and5-hydroxymethyl-2′-deoxycytidine are cytidine derivatives, the presentinventors hypothesized that cells expressing CDA would be resistant totreatment with 5-formyl-2′-deoxycytidine and5-hydroxymethyl-2′-deoxycytidine.

Surprisingly, however, there was in fact no correlation observed betweenCDA expression and resistance or sensitivity to either5-formyl-2′-deoxycytidine or 5-hydroxymethyl-2′-deoxycytidine, as shownin Table 4A. The IC₅₀ data in Table 4 is identical to that in Table 1.CDA expression levels in the cell lines specified were identified usingthe EMBL expression atlas (https://www.ebi.ac.uk/gxa/home, using thesearch term CDA). CDA expression is reported as RNA transcripts permillion (TPM), as described in Wagner et al., (2012) Theory Biosci131(4):281-285 and Mortazavi A et al., (2008) “Mapping and quantifyingmammalian transcriptomes by RNA-Seq.” Nature methods 5(7):621-8. If morethan one value was reported the lowest value was used.

TABLE 4A No correlation between level of CDA expression and sensitivityto treatment with 5-formyl-2′-deoxycytidine or5-hydroxymethyl-2′-deoxycytidine. CDA Cell Germ expression IC₅₀ (μM)Line Disease Layer (TPM) 2d5fC 2d5hmC U87-MG Glioma, Grade IV Ectoderm 60.3340 3.027 HCT-116 Colon Carcinoma Endoderm 24 > >25.00 A549 LungCarcinoma Endoderm 50 > >25.00 22Rv1 Prostate Carcinoma Endoderm1 >25.00 >25.00 NCI-N87 Gastric Carcinoma Endoderm 46 0.8057 >25.00 MIAPancreatic Carcinoma Endoderm 3 1.143 >25.00 A-498 Kidney CarcinomaMesoderm 29 > >25.00 U937 Lymophoma Mesoderm 146 0.9536 >25.00 A375Malignant Melanoma Mesoderm 0 3.188 >25.00 HL-60 Acute PromyelocyticLeukemia Mesoderm 10 6.262 >25.00 SK-OV-3 Ovarian AdenocarcinomaMesoderm 295 9.690 >25.00 MCF-7 Epithelial Adenocarcinoma Mesoderm 00.8657 >25.00 U2OS Bone Osteoscarcoma Mesoderm 13.0 8.69 >25.00 HeLaCervical Adenocarcinoma Mesoderm 42 0.76 >25.00 HAP1 Chronic MyelogenousMesoderm N.A. >25.00 6.53 Leukemia (CML) V79 Chinese Hamster OvaryMesoderm N.A. >25.00 >25.00 H1437 Lung (metastatic non-small Endoderm 495.65 n.d. cell adenocarcinoma) H1573 Lung (metastatic Endoderm 67 7.72n.d. adenocarcinoma) MDA-MB- breast adenocarcinoma, Endoderm 153.0 — —231 HOP-92 non-small cell lung carcinoma Endoderm 321.0 — — Capan-2pancreatic ductal Endoderm 248.0 — — adenocarcinoma

A linear correlation between cytotoxicity and drug exposure showed apoor fit (Linear Model for 2d5fc, R_(z)=0.00173; Linear Model for2d5hmC, R_(z)=0.02262). The data, taken together with the data fromEMBL, suggested that CDA expression is not relevant to5-formyl-2′-deoxycytidine or 5-hydroxymethyl-2′-deoxycytidinesensitivity or resistance.

Additionally, the CDA expression level (TPM) of various glioma celllines was determined, and is shown in Table 4B. CDA expression levels inthe cell lines specified were identified using the Cancer Cell LineEncyclopedia (https://portals.broadinstitute.org/ccle), which isdescribed in Barretina J et al. (2012) The Cancer Cell Line Encyclopediaenables predictive modelling of anticancer drug s8 CDA expression isreported as RNA transcripts per million (TPM), as described in Wagner etal., (2012) Theory Biosci 131(4):281-285. If more than one value wasreported the highest value was used.

TABLE 4B Level of CDA expression in various glioma cell lines. GliomaCell Line CDA expression (TPM) 42-MG-BA 1.0 8-MG-BA 3.0 A172 2.0 AM-387.0 CAS-1 0.2 DBTRG-05MG 0.7 DK-MG 0.1 GAMG 41.0 GB-1 0.5 GMS-10 24.0GOS-3 0.5 KALS-1 24.0 KNS-42 0.4 KNS-60 30.0 KNS-81 0.6 KS-1 7.0 LN-1811.0 LN-229 0.3 M059K 8.0 SF-295 3.0 SF126 16.0 SNB75 0.6 SNU-1105 7.0SNU-201 0.3 SNU-466 0.1 SNU-489 3.0 SNU-626 0.2 T98G 3.0 U-87 MG 6.0YH-13 4.0 YKG1 0.2

Example 8: Non-Mutagenic Effect

A current frontline treatment for GlibolastomaMultiforme—Temozolomide—is potent mutagen. In fact, temozolomide exertsits cancer chemotherapeutic activity is by mutating tumour cell soseverely that the tumour cells are killed. Temozolomide works byalkalyting DNA causing mutations. The expression of MGMT—a proteinresponsible for removing alkalyated DNA damage—makes glioblastoma cellsalmost completely resistant to the cytotoxic effects of Temozolomide.

Therefore, the present inventors investigated whether5-formyl-2′-deoxycytidine and 5-hydroxymethyl-2′-deoxycytidine had asimilar mechanism of action. The mutagenicity of these compounds wasevaluated in an Hypoxanthine-guanine phosphoribosyltransferase (HPRT)assay as described above (Materials and Methods). As shown in the HPRTassay neither 5-hydroxymethyl-2′-deoxycytidine nor5-formyl-2′-deoxycytidine were genotoxic to mammalian cells at anyconcentration evaluated (FIG. 3). These results indicate that the5-hydroxymethyl-2′-deoxycytidine and 5-formyl-2′-deoxycytidine are notmutagens, i.e. that their cytotoxic effects are not due to mutagenicactivity.

Example 9: Cytotoxicity of Other Compounds Vs. HeLa Cells

HeLa cells were treated with either 5-formyl-2′-deoxycytidine,5-formylcytidine or 5-chloro-2′-deoxycytidine. After three days oftreatment with these compounds at a concentration of 100, 25, 6.25,1.56, 0.39, 0.1, 0.02, or 0.006 μM, cell survival was assessed by MTTassay as described above (Materials and Methods).

As shown in FIG. 4. A markedly greater cytotoxic effect is observedfollowing treatment with 5-formyl-2′-deoxycytidine than followingtreatment with either 5-formylcytidine or 5-chloro-2′-deoxycytidine.

Example 10: Cytotoxicity of Other Compounds Vs. Glioma Cells

U87-MG cells (Glioma, Grade IV) were treated with either5-formyl-2′-deoxycytidine, 5-hydroxymethyl-2′-deoxycytidine,5-carboxy-2′-deoxycytidine, Temozolomide, 5-flurouracil,5-bromo-2′-deoxycytidine, 5-iodo-2′-deoxycytidineor5-chloro-2′-deoxycytidine at a concentration of 100, 25, 6.25, 1.56,0.39, 0.1, 0.02, or 0.006 μM. After three days of treatment with thesecompounds, cell survival was assessed by MTT assay as described above(Materials and Methods).

Cell Culture

U87-MG cell lines were grown in DMEM (Sigma, Cat.Nr. D6429) supplementedwith 10% Fetal Bovine Serum. All cells were maintained in a 5% CO2,humidified, water-jacketed incubator at 37° C. Cells were passed between70 and 90% confluence.

Survival Assay

In a 96-well plate, 4000 cells in 100 μl of corresponding medium wereseeded. The following day, drugs, diluted in DMSO, were added intriplicate at a concentration of 100, 25, 6.25, 1.56, 0.39, 0.1, 0.02,or 0.006 μM. Cells were incubated with the drugs for 72 hours. Cellproliferation was assessed by MTT assay according to the manufacturer'sprotocol (ATCC, Cat. Nr. 30-1010K). Cell survival was normalized to thesurvival of cells treated with DMSO only. Experiment was performed threetimes, data represent mean of nine wells±SEM.

As shown in Table 5, both 5-formyl-2′-deoxycytidine and5-hydroxymethyl-2′-deoxycytidine performed surprisingly better in termsof cytotoxicity vs. U87-MG cells than the other compounds tested,including 5-bromo-2′-deoxycytidine, 5-iodo-2′-deoxycytidine and5-chloro-2′-deoxycytidine.

TABLE 5 Evaluation of cytotoxicity of various compounds to U87-MG cells.IC₅₀ is the concentration at which half of the cells are killed by therelevant compound. Cell Line: U87-MG Compound Tested IC₅₀ (μM)5-formyl-2′-deoxycytidine 0.3340 5-hydroxymethyl-2′-deoxycytidine 3.0275-carboxy-2′-deoxycytidine >25.00 Temozolomide >25.005-flurouracil >25.00 5-bromo-2′-deoxycytidine >25.005-chloro-2′-deoxycytidine >25.00 5-iodo-2′-deoxycytidine >25.00

Additionally, U87-MG cells were treated with 5-formyl-2′-deoxycytidine(d5fC), 5-hydroxymethyl-2′-deoxycytidine (d5hmC),5-chloro-2′-deoxycytidine (5CldC), 5-bromo-2′-deoxycytidine (5BrdC),5-lodo-2′deoxycytidine (5IdC), and Thymidine. The results are shown inFIG. 5. d5hmC and d5fC are markedly more cytotoxic than 5CldC, 5BrdC,dldC, and thymidine to glioma cells.

In a 96-well plate, 4000 cells in 100 μl of corresponding medium wereseeded. The following day, drugs, diluted in DMSO, were added intriplicate at a concentration of 100, 25, 6.25, 1.56, 0.39, 0.1, 0.02,or 0.006 μM. Cells were incubated with the drugs for 72 hours. Cellproliferation was assessed by MTT assay according to the manufacturer'sprotocol (ATCC, Cat. Nr. 30-1010K). Cell survival was normalized to thesurvival of cells treated with DMSO only. Experiment was performed threetimes, data represent mean±SD.

Example 11: Effect not Mediated Via Thymidine Synthase Cell Culture

U87-MG cell lines were grown in DMEM (Sigma, Cat.Nr. D6429) supplementedwith 10% Fetal Bovine Serum. All cells were maintained in a 5% CO2,humidified, water-jacketed incubator at 37° C. Cells were passed between70 and 90% confluence.

Survival Assay

In a 96-well plate, 4000 cells in 100 μl of corresponding medium wereseeded. The following day, drugs, diluted in DMSO, were added in 8concentrations in triplicate. Drugs were added as a 4-fold dilutionseries starting from 100 μM. Cells were incubated with the drugs for 72hours. Cell proliferation was assessed by MTT assay according to themanufacturer's protocol (ATCC, Cat. Nr. 30-1010K). Cell survival wasnormalized to the survival of cells treated with DMSO only. Experimentwas performed three times, data represent mean of three wells±SD.

The results are shown in FIG. 6. Cytotoxicity by5-formyl-2′-deoxycytidine and 5-hydroxymethyl-2′-deoxycytidine is notrescued by the addition of thymidine in U87-MG cells. This resultindicates that 5-formyl-2′-deoxycytidine and5-hydroxymethyl-2′-deoxycytidine do not act by inhibiting thymidinesynthase.

Example 12: Combined Therapy with Temozolomide Cell Culture

Glioblastoma multiforme cell lines (U87-MG) were grown in DMEM (Sigma,Cat.Nr. D6429) supplemented with 10% Fetal Bovine Serum. All cells weremaintained in a 5% CO2, humidified, water-jacketed incubator at 37° C.Cells were passed between 70 and 90% confluence.

Survival Assay

In a 96-well plate, 4000 cells in 100 μl of corresponding medium wereseeded. The following day, drugs, diluted in DMSO, were added in 8concentrations in triplicate. Drugs were added as a 4-fold dilutionseries starting from 100 μM. Cells were incubated with the drugs for 72hours. Cell proliferation was assessed by MTT assay according to themanufacturer's protocol (ATCC, Cat. Nr. 30-1010K). Cell survival wasnormalized to the survival of cells treated with DMSO only. Experimentwas performed three times, data represent mean of these experiments.

As shown in FIG. 7, a human glioblastoma multiforme cell line that isresistant to temozolomide was treated with temozolomide alone,5-formyl-2′-deoxycytidine (d5fC) or a combination of d5fC andtemozolomide. The combination of temozolomide and d5fC is more effectiveat treating human glioblastoma multiforme than either chemical alone.

As shown in FIG. 8, a human glioblastoma multiforme cell line that isresistant to temozolomide was treated with temozolomide alone,5-hydroxymethyl-2′-deoxycytidine (d5hmC) or a combination of d5hmC andtemozolomide. The combination of temozolomide and d5hmC is moreeffective at treating human glioblastoma multiforme than either chemicalalone.

Thus, 5-hydroxymethyl-2′-deoxycytidine and 5-formyl-2′-deoxycytidine actsynergistically with temozolomide.

Example 13: Uridine Analogues

The cytotoxic effects of 5-methoxymethyl-2′-deoxyuridine and5-acetoxymethyl-2′-deoxyuridine were evaluated.

Cell Culture

U87-MG cell lines were grown in DMEM (Sigma, Cat.Nr. D6429) supplementedwith 10% Fetal Bovine Serum. All cells were maintained in a 5% CO2,humidified, water-jacketed incubator at 37° C. Cells were passed between70 and 90% confluence.

Survival Assay

In a 96-well plate, 4000 cells in 100 μl of corresponding medium wereseeded. The following day, drugs, diluted in DMSO, were added in 8concentrations in triplicate. Drugs were added as a 4-fold dilutionseries starting from 100 μM. Cells were incubated with the drugs for 72hours. Cell proliferation was assessed by MTT assay according to themanufacturer's protocol (ATCC, Cat. Nr. 30-1010K). Cell survival wasnormalized to the survival of cells treated with DMSO only. Experimentwas performed three times, data represent mean of nine wells±SEM.

As shown in FIG. 9, the U87-MG, glioblastoma multiforme, cell line isunable to survive a treatment with increasing concentrations of5-methoxymethyl-2-deoxyuridine or 5-acetoxymethyl-2′-deoxyuridine.Therefore, 5-methoxymethyl-2-deoxyuridine and5-acetoxymethyl-2′-deoxyuridine are effective anti-cancer agents,particularly against glioblastoma multiforme.

Example 14: Cytotoxicity of 5-Formyl-2′-Deoxycytidine-5′-Triphosphate(HeLa) Cell Culture

HeLa cell lines were grown in DMEM (Sigma, Cat.Nr. D6429) supplementedwith 10% Fetal Bovine Serum. All cells were maintained in a 5% CO₂,humidified, water-jacketed incubator at 37° C. Cells were passed between70 and 90% confluence.

Survival Assay

In a 96-well plate, 4000 cells in 100 μl of corresponding medium wereseeded. The following day, drugs, diluted in DMSO, were added in 8concentrations in triplicate. Drugs were added as a 4-fold dilutionseries starting from 100 μM. Cells were incubated with the drugs for 72hours. Cell proliferation was assessed by MTT assay according to themanufacturer's protocol (ATCC, Cat. Nr. 30-1010K). Cell survival wasnormalized to the survival of cells treated with DMSO only. Experimentwas performed three times, data represent mean of nine wells±SEM.

As shown in FIG. 10A, HeLa cervical carcinoma cells are unable tosurvive a treatment with increasing concentrations of5-formyl-2′-deoxycytidine-5′-triphosphate. This demonstrates5-formyl-2′-deoxycytidine-5′-triphosphate's utility in the treatment ofhuman cancers, for instance cervical carcinoma.

Example 15: Cytotoxicity of 5-Formyl-2′-Deoxycytidine-5′-Triphosphateand 5-Hydroxymethyl-2′-Deoxycytidine-5′-Triphosphate (Glioma)

U87-MG cells (Glioma, Grade IV) were treated with either5-formyl-2′-deoxycytidine-5′-triphosphate or5-hydroxymethyl-2′-deoxycytidine-5′-triphosphate at a concentration of100, 25, 6.25, 1.56, 0.39, 0.1, 0.02, or 0.006 μM. After three days oftreatment with these compounds, cell survival was assessed by MTT assayas described above (Materials and Methods).

Cell Culture

U87-MG cell lines were grown in DMEM (Sigma, Cat.Nr. D6429) supplementedwith 10% Fetal Bovine Serum. All cells were maintained in a 5% CO2,humidified, water-jacketed incubator at 37° C. Cells were passed between70 and 90% confluence.

Survival Assay

In a 96-well plate, 4000 cells in 100 μl of corresponding medium wereseeded. The following day, drugs, diluted in DMSO, were added intriplicate at a concentration of 100, 25, 6.25, 1.56, 0.39, 0.1, 0.02,or 0.006 μM. Cells were incubated with the drugs for 72 hours. Cellproliferation was assessed by MTT assay according to the manufacturer'sprotocol (ATCC, Cat. Nr. 30-1010K). Cell survival was normalized to thesurvival of cells treated with DMSO only. Experiment was performed threetimes, data represent mean of at least 3 wells±SD.

As shown in FIG. 10B, both 5-formyl-2′-deoxycytidine-5′-triphosphate and5-hydroxymethyl-2′-deoxycytidine-5′-triphosphate were cytotoxic toU87-MG, glioma, cells.

Example 16: CDA Expression

The linear and logarithm base 2 transformed CDA expression level ofvarious glioma cell lines was determined. CDA expression levels in thecell lines specified were identified using the GENEVESTIGATOR® database(https://genevestigator.com/gv/), by searching for CDA, organism:homosapiens, platform: Affymetrix Human Genome U133 2.0 Array. Theresults are shown in FIGS. 11 to 13 and Table 6.

FIG. 11 shows the CDA normalized Log 2 CDA expression levels determinedfor various cancers. FIG. 12 shows the linear CDA expression levelsdetermined for various cancers. FIGS. 13A and 13B show the CDAnormalized Log 2 expression levels in various human brain tumours. FIGS.14A and 14B show the linear CDA expression levels in various human braintumours.

Table 6 below shows the CDA expression levels of various cell lines andthe IC₅₀ values for 2d5hmC and/or 2d5fc in those cell lines. IC₅₀ valueswere obtained as described above (Materials and Methods and Example 1).

TABLE 6 CDA expression levels (Log2 normalized values) and 2d5hmC and/or2d5fc IC₅₀ values in various cell lines. CDA CDA Expression IC50 IC50CDA Mean Expression Median Cell Line 2d5hmC 2d5fC Log2 SD Log2 Log2U87-MG 3.027 0.334 9.84 0.42 9.94 HCT-116 25 25 11.11 0.62 10.93 A549 2525 12 1.01 11.93 22Rv1 25 25 8.92 0.62 9.05 NCI-N87 25 0.8057 11.6 0.5611.37 MIA PaCa-2 25 1.143 9.68 0.49 9.79 A-498 25 25 10.52 1.06 10.32U937 25 0.9536 13.82 0.45 13.89 A375 25 3.188 9.36 0.62 9.56 HL-60 256.262 10.36 0.96 10.12 SK-OV-3 25 9.69 13.35 1.59 14.07 MCF-7 25 0.86579.26 0.39 9.2 U2OS 25 8.69 10.14 10.14 HeLa 25 0.76 13.15 0.92 13.33HAP1 6.53 25 None HEK293 25 25 9.59 0.4 9.53 ARPE19 25 25 9.99 0.7 9.94MRC5 25 25 9.87 0.29 9.82 H1437 — 5.65 13.02 1.12 12.44 H1573 — 7.7213.63 0.18 13.67Table 7 below shows the linear CDA expression levels of various celllines and the IC₅₀ values for 2d5hmC and/or 2d5fc in those cell lines.IC₅₀ values were obtained as described above (Materials and Methods andExample 1).

TABLE 7 Linear CDA expression levels and 2d5hmC and/or 2d5fc IC₅₀ valuesin various cell lines. CDA CDA IC50 IC50 Linear CDA Linear Cell Line2d5hmC 2d5fC Mean Linear SD Median U87-MG 3.027 0.334 951.65 276.02989.44 HCT-116 25 25 2481.67 1559.27 1949.23 A549 25 25 5490.76 5549.263904.87 22Rv1 25 25 519.39 225.08 527.89 NCI-N87 25 0.8057 3327.031464.72 2654.16 MIA PaCa-2 25 1.143 860.01 261.17 881.38 A-498 25 251874.54 1443.37 1301.02 U937 25 0.9536 15148.69 4611.68 15231.25 A375 253.188 700.89 226.39 753.10 HL-60 25 6.262 1787.26 2129.78 1112.79SK-OV-3 25 9.69 15500.02 11439.83 17236.82 MCF-7 25 0.8657 635.57 182.06588.96 U2OS 25 8.69 1130.20 N/A 1130.20 HeLa 25 0.76 10915.38 6549.9510272.63 HAP1 6.53 25 *** *** *** HEK293 25 25 800.57 225.25 738.45ARPE19 25 25 1133.42 515.09 981.92 MRC5 25 25 950.82 212.00 901.07 H1437— 5.65 11861.95 14192.00 5568.28 H1573 — 7.72 12779.53 1515.78 13045.67Together these results demonstrate that many cancers, including alltested cancers of the central nervous system, do not over-express CDA;rather they express low levels of CDA. The results also demonstrate thatthere is no correlation between CDA expression and sensitivity to either5-formyl-2′-deoxycytidine or 5-hydroxymethyl-2′-deoxycytidine

Example 17: Blood-Brain Barrier Permeability

Blood brain barrier permeability was measured using a ParallelArtificial Membrane Permeability Assay Kit (PAMPA) from BioAssaysystems. The manufacturer's instructions were followed to determinepermeability.

Manufacturer's instructions: 1. In separate centrifuge tubes, prepare500 μL of 500 μM Test Compound: mix 25 μL 10 mM Test Compound inDMSO+475 μL PBS. If using the Permeability Controls, dilute them to 500μM as well: mix 25 μL Permeability Control+475 μL PBS. 2. In separatetubes, prepare 200 μM Equilibrium Standards for each test compound andcontrol: mix 80 μL of 500 μM Test Compound or Control with 120 μL PBS.If the compound is able to permeabilize the membrane and fully reachequilibrium, 200 μM will be the final concentration of solution in theDonor and Acceptor wells. Next, in a separate tube, mix 5 μL DMSO+245 μLPBS to prepare the Blank Control. Set aside the Equilibrium Standardsand Blank Control for analysis the next day. 3. Add 300 μL PBS to wellsin the acceptor plate. 4. With the donor plate still in its tray, add 5μL 4% Lecithin in Dodecane directly to the well membranes of the donorplate. Be careful not to puncture the membranes with the pipette tip. 5.Add 200 μL of each 500 μM Test Compound and 500 μM Permeability Controlsto duplicate wells of the donor plate. Note: we recommend running allexperimental variables in at least duplicate5. Carefully place the donorplate into the acceptor plate wells. Incubate at RT or 37° C. for 18hours or the desired incubation time period (e.g. 16-24 hours) 6.Carefully remove donor plate and collect the liquid in acceptor platewells for analysis. This will be referred to as Acceptor Solution 7. Add100 μL of Acceptor Solution and Equilibrium Standards for each TestCompound and Permeability Control. Also add 100 μL Blank Control towells of UV plate (Cat # P96UV). 8. Read Absorbance spectrum from 200 nmto 500 nm in 10 nm intervals to determine peak absorbance of testcompounds. The Blank Control is to confirm peaks are due to the testcompound and not the DMSO in the solution. Peak absorbance for HighPermeability, Medium Permeability, and Low Permeability Controls are 280nm, 270 nm, and 270 nm respectively.

As shown in FIG. 15, 2d5hmC and 2d5fC pass the blood brain barrier.

1. A method of treating or preventing cancer in a subject, whichcomprises administering to the subject a compound of Formula (I):

or a stereoisomer, solvate, tautomer or pharmaceutically acceptable saltthereof, wherein: X is a group containing from 1 to 20 non-hydrogenatoms, which contains at least one functional group selected from thegroup consisting of: an aldehyde, an alcohol, a protected alcohol, anether, an ester, and a carboxylic acid, with the proviso that X is not—COOH; W₁ and W₂ are each independently O, S, or NH; Y is H or a groupcontaining from 1 to 15 non-hydrogen atoms; Z is —OPG, —OR_(z) or—N(R_(x)R_(y)), where R_(x), R_(y), and R_(z) are independently H or agroup containing from 1 to 10 non-hydrogen atoms; R₁ is H or a groupcontaining from 1 to 15 non-hydrogen atoms; R₂ is H, —OH, —OPG, —F, —Cl,—Br, —I, or —N₃; R₃ is H, —F, —Cl, —Br, —I, or —N₃; and PG is an alcoholprotecting group.
 2. The method of claim 1, wherein PG is acetyl (Ac),benzyl (Bn), or benzoyl (Bz).
 3. The method of claim 1, wherein thecompound is a compound of Formula (IIa) or Formula (IIb), or astereoisomer, solvate, tautomer, or pharmaceutically acceptable saltthereof:

wherein X, R₁,and R₂ are as defined in claim
 1. 4. The method of claim1, wherein the compound is a compound of Formula (IIIa), (IIIb), (IIIc),or (IIId), or a stereoisomer, solvate, tautomer, or pharmaceuticallyacceptable salt thereof:

wherein X is as defined in claim
 1. 5. The method of claim 1, wherein: Xis —(CH₂)_(n)—X′, n is from 0 to 6, X′ is —CHO, —OH, —OR, or —OC(═O)R,and R is methyl.
 6. The method of claim 1, wherein X is a groupcomprising from 2 to 20 non-hydrogen atoms.
 7. The method of claim 1,wherein X is not —COOH or —OH.
 8. The method of claim 1, wherein X is:a) —CHO or —CH₂OH; or b) —CH₂OCH₃ or —CH₂OC(═O)CH₃.
 9. The method ofclaim 1, wherein X is —CHO or —CH₂OH.
 10. The method of claim 1, whereinX is CH₂OH.
 11. The method of claim 1, wherein Z is —NH₂.
 12. The methodof claim 1, wherein the compound is 5-formyl-2′-deoxycytidine,5-hydroxymethyl-2′-deoxycytidine, 5-methoxymethyl-2′-deoxyuridine,5-acetoxymethyl-2′-deoxyuridine,5-formyl-2′-deoxycytidine-5′-triphosphate or5-hydroxymethyl-2′-deoxycytidine-5′-triphosphate, or a stereoisomer,solvate, tautomer, or pharmaceutically acceptable salt thereof.
 13. Themethod of claim 12, wherein the compound is 5-formyl-2′-deoxycytidine or5-hydroxymethyl-2′-deoxycytidine, or a stereoisomer, solvate, tautomer,or pharmaceutically acceptable salt thereof,
 14. The method of claim 13,wherein the compound is 5-hydroxymethyl-2′-deoxycytidine or astereoisomer, solvate, tautomer, or pharmaceutically acceptable saltthereof.
 15. The method of claim 1, wherein the cancer is an ectoderm,paraxial mesoderm, or lateral plate mesoderm cancer.
 16. The method ofclaim 1, wherein the cancer is a central nervous system cancer.
 17. Themethod of claim 1, wherein the cancer is a glioma.
 18. The method ofclaim 1, wherein cytidine deaminase (CDA) is not over-expressed in thecancer.
 19. The method of claim 18, wherein the CDA expression level inthe cancer is not greater than 90% of the CDA expression level in areference cancer cell line as determined using the same method under thesame conditions, wherein said reference cancer cell line is MDA-MB-231.20. The method of claim 1, wherein the cancer is resistant to treatmentwith gemcitabine, cytarabine, temozolomide, or 5-fluorouracil.
 21. Themethod of claim 1, wherein the compound is administered at a dose ofbetween 10 mg/kg and 405 mg/kg.
 22. The method of claim 1, furthercomprising administering an additional anticancer agent.
 23. A kitcomprising a compound of Formula (I):

or a stereoisomer, solvate, tautomer or pharmaceutically acceptable saltthereof, wherein: X is a group containing from 1 to 20 non-hydrogenatoms, which contains at least one functional group selected from thegroup consisting of: an aldehyde, an alcohol, a protected alcohol, anether, an ester, and a carboxylic acid, with the proviso that X is not—COOH; W₁ and W₂ are each independently 0, S, or NH; Y is H or a groupcontaining from 1 to 15 non-hydrogen atoms; Z is —OPG, —OR_(z) or—N(R_(x)R_(y)), where R_(x), R_(X), and R_(z) are independently H or agroup containing from 1 to 10 non-hydrogen atoms; R₁ is H or a groupcontaining from 1 to 15 non-hydrogen atoms; R₂ is H, —OH, —OPG, —F, —Cl,—Br, —I, or —N₃; R₃ is H, —F, —Cl, —Br, —I, or —N₃; and PG is an alcoholprotecting group, and an additional anticancer agent.
 24. Apharmaceutical composition comprising a compound of Formula (I):

or a stereoisomer, solvate, tautomer or pharmaceutically acceptable saltthereof, wherein: X is a group containing from 1 to 20 non-hydrogenatoms, which contains at least one functional group selected from thegroup consisting of: an aldehyde, an alcohol, a protected alcohol, anether, an ester, and a carboxylic acid, with the proviso that X is not—COOH; W₁ and W₂ are each independently O, S, or NH; Y is H or a groupcontaining from 1 to 15 non-hydrogen atoms; Z is —OPG, —OR_(z) or—N(R_(x)R_(y)), where R_(x), R_(y), and R_(z) are independently H or agroup containing from 1 to 10 non-hydrogen atoms; R₁ is H or a groupcontaining from 1 to 15 non-hydrogen atoms; R₂ is H, —OH, —OPG, —F, —Cl,—Br, —I, or —N₃; R₃ is H, —F, —Cl, —Br, —I, or —N₃; and PG is an alcoholprotecting group, and one or more pharmaceutically acceptableexcipients.
 25. The kit of claim 23, wherein the additional anticanceragent is selected from the group consisting of: gemcitabine, cytarabine,temozolomide, 5-fluorouracil, and carmustine.
 26. (canceled)
 27. Themethod of claim 3, wherein the compound is a compound of Formula (IIa).28. The method of claim 4, wherein the compound is a compound of Formula(IIIa) or Formula (IIIc).
 29. The method of claim 16, wherein the canceris a brain cancer.
 30. The method of claim 17, wherein the cancer is aglioblastoma.
 31. The method of claim 18, wherein the cancer expressesCDA at a level of ≤140 transcripts per million (TPM).
 32. The method ofclaim 22, wherein the additional anticancer agent is selected from thegroup consisting of: gemcitabine, cytarabine, temozolomide,5-fluorouracil, and carmustine.
 33. The composition of claim 24, furthercomprising an additional anticancer agent.
 34. The composition of claim34, wherein the additional anticancer agent is selected from the groupconsisting of: gemcitabine, cytarabine, temozolomide, 5-fluorouracil,and carmustine.